Antibodies against a secreted polypeptide that stimulates release of proteoglycans from cartilage

ABSTRACT

The present invention is directed to secreted and transmembrane polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 09/866,028 filed May 25, 2001, whichis a continuation of, and claims priority under 35 USC §120 to, PCTApplication PCT/US99/28301 filed Dec. 1, 1999, which is acontinuation-in-part of, and claims priority under 35 USC §120 to, U.S.application Ser. No. 09/254,311 filed Mar. 3, 1999, now abandoned, whichis the National Stage filed under 35 USC §371 of PCT ApplicationPCT/US98/25108 filed Dec. 1, 1998, which claims priority under 35 USC§119 to U.S. Provisional Application No. 60/069,334 filed Dec. 11, 1997.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

1. PRO241

Cartilage is a specialized connective tissue with a large extracellularmatrix containing a dense network of collagen fibers and a high contentof proteoglycan. While the majority of the proteoglycan in cartilage isaggrecan, which contains many chondroitin sulphate and keratin sulphatechains and forms multimolecular aggregates by binding with link proteinto hyaluronan, cartilage also contains a number of smaller molecularweight proteoglycans. One of these smaller molecular weightproteoglycans is a protein called biglycan, a proteoglycan which iswidely distributed in the extracellular matrix of various otherconnective tissues including tendon, sclera, skin, and the like.Biglycan is known to possess leucine-rich repeat sequences and twochondroitin sulphate/dermatan sulphate chains and functions to bind tothe cell-binding domain of fibronectin so as to inhibit cellularattachment thereto. It is speculated that the small molecular weightproteoglycans such as biglycan may play important roles in the growthand/or repair of cartilage and in degenrative diseases such asarthritis. As such, there is an interest in identifying andcharacterizing novel polypeptides having homology to biglycan protein.

We herein describe the identification and characterization of novelpolypeptides having homology to the biglycan protein, wherein thosepolypeptides are herein designated PRO241 polypeptides.

2. PRO243

Chordin (Xenopus, Xchd) is a soluble factor secreted by the Spemannorganizer which has potent dorsalizing activity (Sasai et al., Cell 79:779-90 (1994); Sasai et al, Nature 376: 333-36 (1995). Other dorsalizingfactors secreted by the organizer are noggin (Smith and Harlan, Cell 70:829-840 (1992); Lamb et al, Science 262: 713-718 (1993) and follistatin(Hemmanti-Brivanlou et al, Cell 77: 283-295 (1994). Chordin subdividesprimitive ectoderm into neural versus non-neural domains, and inducesnotochord and muscle formation by the dorsalization of the mesoderm. Itdoes this by functioning as an antagonist of the ventralizing BMP-4signals. This inhibition is mediated by direct binding of chordin toBMP-4 in the extracellular space, thereby preventing BMP-4 receptoractivation by BMP4 (Piccolo et al., Develop. Biol. 182: 5-20 (1996).

BMP4 is expressed in a gradient from the ventral side of the embryo,while chordin is expressed in a gradient complementary to that of BMP-4.Chordin antagonizes BMP-4 to establish the low end of the BMP4 gradient.Thus, the balance between the signal from chordin and otherorganizer-derived factors versus the BMP signal provides the ectodermalgerm layer with its dorsal-ventral positional information. Chordin mayalso be involved in the dorsal-ventral patterning of the central nervoussystem (Sasai et al, Cell 79: 779-90 (1994). It also induces exclusivelyanterior neural tissues (forebrain-type), thereby anteriorizing theneural type (Sasai et al, Cell 79: 779-90 (1997). Given its role inneuronal induction and patterning, chordin may prove useful in thetreatment of neurodegenerative disorders and neural damage, e.g., due totrauma or after chemotherapy.

We herein describe the identification and characterization of novelpolypeptides having homology to the chordin protein, wherein thosepolypeptides are herein designated PRO243 polypeptides.

3. PRO299

The notch proteins are involved in signaling during development. Theymay effect asymmetric development potential and may signal expression ofother proteins involved in development. [See Robey, E., Curr. Opin.Genet. Dev., 7(4):551 (1997), Simpson, P., Curr. Opin. Genet. Dev.,7(4):537 (1997), Blobel, CP., Cell, 90(4):589 (1997)], Nakayama, H. etal., Dev. Genet., 21(1):21 (1997), Nakayama, H. et al., Dev. Genet.,21(1):21 (1997), Sullivan, S. A. et al., Dev. Genet., 20(3):208 (1997)and Hayashi, H. et al., Int. J. Dev. Biol., 40(6):1089 (1996).]Serrate-mediated activation of notch has been observed in the dorsalcompartment of the Drosophila wing imaginal disc. Fleming et al.,Development, 124(15):2973 (1997). Notch is of interest for both its rolein development as well as its signaling abilities. Also of interest arenovel polypeptides which may have a role in development and/orsignaling.

We herein describe the identification and characterization of novelpolypeptides having homology to the notch protein, wherein thosepolypeptides are herein designated PRO299 polypeptides.

4. PRO323

Dipeptidases are enzymatic proteins which function to cleave a largevariety of different dipeptides and which are involved in an enormousnumber of very important biological processes in mammalian andnon-mammalian organisms. Numerous different dipeptidase enzymes from avariety of different mammalian and non-mammalian organisms have beenboth identified and characterized. The mammalian dipeptidase enzymesplay important roles in many different biological processes including,for example, protein digestion, activation, inactivation, or modulationof dipeptide hormone activity, and alteration of the physical propertiesof proteins and enzymes.

In light of the important physiological roles played by dipeptidaseenzymes, efforts are being undertaken by both industry and academia toidentify new, native dipeptidase homologs. Many efforts are focused onthe screening of mammalian recombinant DNA libraries to identify thecoding sequences for novel secreted and membrane-bound receptorproteins. Examples of screening methods and techniques are described inthe literature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having homology to various dipeptidase enzymes, designatedherein as PRO323 polypeptides.

5. PRO327

The anterior pituitary hormone prolactin is encoded by a member of thegrowth hormone/prolactin/placental lactogen gene family. In mammals,prolactin is primarily responsible for the development of the mammarygland and lactation. Prolactin functions to stimulate the expression ofmilk protein genes by increasing both gene transcription and mRNAhalf-life.

The physiological effects of the prolactin protein are mediated throughthe ability of prolactin to bind to a cell surface prolactin receptor.The prolactin receptor is found in a variety of different cell types,has a molecular mass of approximately 40,000 and is apparently notlinked by disulfide bonds to itself or to other subunits. Prolactinreceptor levels are differentially regulated depending upon the tissuestudied.

Given the important physiological roles played by cell surface receptormolecules in vivo, efforts are currently being undertaken by bothindustry and academia to identify new, native membrane-bound receptorproteins, including those which share sequence homology with theprolactin receptor. Many of these efforts are focused on the screeningof mammalian recombinant DNA libraries to identify the coding sequencesfor novel membrane-bound receptor proteins. Examples of screeningmethods and techniques are described in the literature [see, forexample, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S.Pat. No. 5,536,637)].

We herein describe the identification and characterization of novelpolypeptides having significant homology to the prolactin receptorprotein, designated herein as PRO327 polypeptides.

6. PRO233

Studies have reported that the redox state of the cell is an importantdeterminant of the fate of the cell. Furthermore, reactive oxygenspecies have been reported to be cytotoxic, causing inflammatorydisease, including tissue necrosis, organ failure, atherosclerosis,infertility, birth defects, premature aging, mutations and malignancy.Thus, the control of oxidation and reduction is important for a numberof reasons, including the control and prevention of strokes, heartattacks, oxidative stress and hypertension.

Oxygen free radicals and antioxidants appear to play an important rolein the central nervous system after cerebral ischemia and reperfusion.Moreover, cardiac injury, related to ischemia and reperfusion has beenreported to be caused by the action of free radicals. In this regard,reductases, and particularly, oxidoreductases, are of interest. Inaddition, the transcription factors, NF-kappa B and AP-1, are known tobe regulated by redox state and to affect the expression of a largevariety of genes thought to be involved in the pathogenesis of AIDS,cancer, atherosclerosis and diabetic complications. Publications furtherdescribing this subject matter include Kelsey et al., Br. J. Cancer,76(7):852-854 (1997); Friedrich and Weiss, J. Theor. Biol.,187(4):529-540 (1997) and Pieulle et al., J. Bacteriol.,179(18):5684-5692 (1997). Given the physiological importance of redoxreactions in vivo, efforts are currently being under taken to identifynew, native proteins which are involved in redox reactions. We describeherein the identification and characterization of novel polypeptideswhich have homology to reductase, designated herein as PRO233polypeptides.

7. PRO344

The complement proteins comprise a large group of serum proteins some ofwhich act in an enzymatic cascade, producing effector molecules involvedin inflammation. The complement proteins are of particular physiologicalimportance in regulating movement and function of cells involved ininflammation. Given the physiological importance of inflammation andrelated mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in inflamation. Wedescribe herein the identification and characterization of novelpolypeptides which have homology to complement proteins, wherein thosepolypeptides are herein designated as PRO344 polypeptides.

8. PRO347

Cysteine-rich proteins are generally proteins which have intricatethree-dimensional structures and/or exist in multimeric forms due to thepresence of numerous cysteine residues which are capable of formingdisulfide bridges. One well known cysteine-rich protein is the mannosereceptor which is expressed in, among other tissues, liver where itserves to bind to mannose and transport it into liver cells. Othercysteine-rich proteins are known to play important roles in many otherphysiological and biochemical processes. As such, there is an interestin identifying novel cysteine-rich proteins. In this regard, Applicantsdescribe herein the identification and characterization of novelcysteine-rich polypeptides that has significant sequence homology to thecysteine-rich secretory protein-3, designated herein as PRO347polypeptides.

9. PRO354

Inter-alpha-trypsin inhibitor (ITI) is a large (Mr approximately240,000) circulating protease inhibitor found in the plasma of manymammalian species. The intact inhibitor is a glycoprotein and consistsof three glycosylated subunits that interact through a strongglycosaminoglycan linkage. The anti-trypsin activity of ITI is locatedon the smallest subunit (i.e., the light chain) of the complex, whereinthat light chain is now known as the protein bikunin. The mature lightchain consists of a 21-amino acid N-terminal sequence, glycosylated atSer-10, followed by two tandem Kunitz-type domains, the first of whichis glycosylated at Asn-45 and the second of which is capable ofinhibiting trypsin, chymotrypsin and plasmin. The remaining two chainsof the ITI complex are heavy chains which function to interact with theenzymatically active light chain of the complex.

Efforts are being undertaken by both industry and academia to identifynew, native proteins. Many efforts are focused on the screening ofmammalian recombinant DNA libraries to identify the coding sequences fornovel secreted and membrane-bound receptor proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)]. We herein describe the identification andcharacterization of novel polypeptides having significant homology tothe ITI heavy chain, designated in the present application as PRO354polypeptides.

10. PRO355

Cytotoxic or regulatory T cell associated molecule or “CRTAM” protein isstructurally related to the immunoglobulin superfamily. The CRTAMprotein should be capable of mediating various immune responses.Antibodies typically bind to CRTAM proteins with high affinity. Zlotnik,A., Faseb, 10(6): A1037, Abr. 216, June 1996. Given the physiologicalimportance of T cell antigens and immune processes in vivo, efforts arecurrently being under taken to identify new, native proteins which areinvolved in immune responses. See also Kennedy et al., U.S. Pat. No.5,686,257 (1997). We describe herein the identification andcharacterization of novel polypeptides which have homology to CRTAM,designated in the present application as PRO355 polypeptides.

11. PRO357

Protein-protein interactions include receptor and antigen complexes andsignaling mechanisms. As more is known about the structural andfunctional mechanisms underlying protein-protein interactions,protein-protein interactions can be more easily manipulated to regulatethe particular result of the protein-protein interaction. Thus, theunderlying mechanisms of protein-protein interactions are of interest tothe scientific and medical community.

All proteins containing leucine-rich repeats are thought to be involvedin protein-protein interactions. Leucine-rich repeats are short sequencemotifs present in a number of proteins with diverse functions andcellular locations. The crystal structure of ribonuclease inhibitorprotein has revealed that leucine-rich repeats correspond to beta-alphastructural units. These units are arranged so that they form a parallelbeta-sheet with one surface exposed to solvent, so that the proteinacquires an unusual, nonglobular shape. These two features have beenindicated as responsible for the protein-binding functions of proteinscontaining leucine-rich repeats. See, Kobe and Deisenhofer, TrendsBiochem. Sci., 19(10):415-421 (October 1994).

A study has been reported on leucine-rich proteoglycans which serve astissue organizers, orienting and ordering collagen fibrils duringontogeny and are involved in pathological processes such as woundhealing, tissue repair, and tumor stroma formation. Iozzo, R. V., Crit.Rev. Biochem. Mol. Biol., 32(2):141-174 (1997). Others studiesimplicating leucine rich proteins in wound healing and tissue repair areDe La Salle, C., et al., Vouv. Rev. Fr. Hematol. (Germany),37(4):215-222 (1995), reporting mutations in the leucine rich motif in acomplex associated with the bleeding disorder Bernard-Soulier syndrome,Chlemetson, K. J., Thromb. Haemost. (Germany), 74(1): 111-116 (July1995), reporting that platelets have leucine rich repeats and Ruoslahti,E. I., et al., WO9110727-A by La Jolla Cancer Research Foundationreporting that decorin binding to transforming growth factors hasinvolvement in a treatment for cancer, wound healing and scarring.Related by function to this group of proteins is the insulin like growthfactor (IGF), in that it is useful in wound-healing and associatedtherapies concerned with re-growth of tissue, such as connective tissue,skin and bone; in promoting body growth in humans and animals; and instimulating other growth-related processes. The acid labile subunit(ALS) of IGF is also of interest in that it increases the half-life ofIGF and is part of the IGF complex in vivo.

Another protein which has been reported to have leucine-rich repeats isthe SLIT protein which has been reported to be useful in treatingneuro-degenerative diseases such as Alzheimer's disease, nerve damagesuch as in Parkinson's disease, and for diagnosis of cancer, see,Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by YaleUniversity. Also of interest is LIG-1, a membrane glycoprotein that isexpressed specifically in glial cells in the mouse brain, and hasleucine rich repeats and immunoglobulin-like domains. Suzuki, et al., J.Biol. Chem. (U.S.), 271(37):22522 (1996). Other studies reporting on thebiological functions of proteins having leucine rich repeats include:Tayar, N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70(December 1996) (gonadotropin receptor involvement); Miura, Y., et al.,Nipon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosisinvolvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,6(4):1125-1133 (October 1995) (kidney disease involvement).

Efforts are therefore being undertaken by both industry and academia toidentify new proteins having leucine rich repeats to better understandprotein-protein interactions. Of particular interest are those proteinshaving leucine rich repeats and homology to known proteins havingleucine rich repeats such as the acid labile subunit of insulin-likegrowth factor. Many efforts are focused on the screening of mammalianrecombinant DNA libraries to identify the coding sequences for novelsecreted and membrane-bound proteins having leucine rich repeats.Examples of screening methods and techniques are described in theliterature [see, for example, Klein et al., Proc. Natl. Acad. Sci.,93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].

We describe herein the identification and characterization of novelpolypeptides having homology to the acid labile subunit of insulin-likegrowth factor, designated in the present application as PRO357polypeptides.

12. PRO715

Control of cell numbers in mammals is believed to be determined, inpart, by a balance between cell proliferation and cell death. One formof cell death, sometimes referred to as necrotic cell death, istypically characterized as a pathologic form of cell death resultingfrom some trauma or cellular injury. In contrast, there is another,“physiologic” form of cell death which usually proceeds in an orderly orcontrolled manner. This orderly or controlled form of cell death isoften referred to as “apoptosis” [see, e.g., Barr et al.,Bio/Technology. 12:487-493 (1994); Steller et al., Science,267:1445-1449 (1995)]. Apoptotic cell death naturally occurs in manyphysiological processes, including embryonic development and clonalselection in the immune system [Itoh et al., Cell, 66:233-243 (1991)].Decreased levels of apoptotic cell death have been associated with avariety of pathological conditions, including cancer, lupus, and herpesvirus infection [Thompson, Science, 267:1456-1462 (1995)]. Increasedlevels of apoptotic cell death may be associated with a variety of otherpathological conditions, including AIDS, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis,retinitis pigmentosa, cerebellar degeneration, aplastic anemia,myocardial infarction, stroke, reperfusion injury, and toxin-inducedliver disease [see, Thompson, supra].

Apoptotic cell death is typically accompanied by one or morecharacteristic morphological and biochemical changes in cells, such ascondensation of cytoplasm, loss of plasma membrane microvilli,segmentation of the nucleus, degradation of chromosomal DNA or loss ofmitochondrial function. A variety of extrinsic and intrinsic signals arebelieved to trigger or induce such morphological and biochemicalcellular changes [Raff, Nature, 356:397-400 (1992); Steller, supra;Sachs et al., Blood, 82:15 (1993)]. For instance, they can be triggeredby hormonal stimuli, such as glucocorticoid hormones for immaturethymocytes, as well as withdrawal of certain growth factors[Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)]. Also, someidentified oncogenes such as myc, rel, and E1A, and tumor suppressors,like p53, have been reported to have a role in inducing apoptosis.Certain chemotherapy drugs and some forms of radiation have likewisebeen observed to have apoptosis-inducing activity [Thompson, supra].

Various molecules, such as tumor necrosis factor-α” (“TNF-α”), tumornecrosis factor-β (“TNF-β” or “lymphotoxin-α”), lymphotoxin-β (“LT-β”),CD30 ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1ligand (also referred to as Fas ligand or CD95 ligand), and Apo-2 ligand(also referred to as TRAIL) have been identified as members of the tumornecrosis factor (“TNF”) family of cytokines [See, e.g., Gruss and Dower,Blood, 85:3378-3404 (1995); Pitti et al., J. Biol. Chem.,271:12687-12690 (1996); Wiley et al., Immunity, 3:673-682 (1995);Browning et al., Cell, 72:847-856 (1993); Armitage et al. Nature,357:80-82 (1992)]. Among these molecules, TNF-α, TNF-β, CD30 ligand,4-1BB ligand, Apo-1 ligand, and Apo-2 ligand (TRAIL) have been reportedto be involved in apoptotic cell death. Both TNF-α and TNF-β have beenreported to induce apoptotic death in susceptible tumor cells [Schmid etal., Proc. Natl. Acad. Sci., 83:1881 (1986); Dealtry et al., Eur. J.Immunol., 17:689 (1987)]. Zheng et al. have reported that TNF-α isinvolved in post-stimulation apoptosis of CD8-positive T cells [Zheng etal., Nature, 377:348-351 (1995)]. Other investigators have reported thatCD30 ligand may be involved in deletion of self-reactive T cells in thethymus [Amakawa et al., Cold Spring Harbor Laboratory Symposium onProgrammed Cell Death, Abstr. No. 10, (1995)].

Mutations in the mouse Fas/Apo-1 receptor or ligand genes (called lprand gld, respectively) have been associated with some autoimmunedisorders, indicating that Apo-1 ligand may play a role in regulatingthe clonal deletion of self-reactive lymphocytes in the periphery[Krammer et al., Curr. Op. Immunol., 6:279-289 (1994); Nagata et al.,Science, 267:1449-1456 (1995)]. Apo-1 ligand is also reported to inducepost-stimulation apoptosis in CD4-positive T lymphocytes and in Blymphocytes, and may be involved in the elimination of activatedlymphocytes when their function is no longer needed [Krammer et al.,supra; Nagata et al., supra]. Agonist mouse monoclonal antibodiesspecifically binding to the Apo-1 receptor have been reported to exhibitcell killing activity that is comparable to or similar to that of TNF-α[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].

Induction of various cellular responses mediated by such TNF familycytokines is believed to be initiated by their binding to specific cellreceptors. Two distinct TNF receptors of approximately 55-kDa (TNFR1)and 75-kDa (TNFR2) have been identified [Hohman et al., J. Biol. Chem.,264:14927-14934 (1989); Brockhaus et al., Proc. Natl. Acad. Sci.,87:3127-3131 (1990); EP 417,563, published Mar. 20, 1991] and human andmouse cDNAs corresponding to both receptor types have been isolated andcharacterized [Loetscher et al., Cell, 61:351 (1990); Schall et al.,Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023 (1990); Lewiset al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991); Goodwin et al.,Mol. Cell. Biol., 11:3020-3026 (1991)]. The TNF family ligandsidentified to date, with the exception of lymphotoxin-α, are type IItransmembrane proteins, whose C-terminus is extracellular. In contrast,most receptors in the TNF receptor (TNFR) family identified to date aretype I transmembrane proteins. In both the TNF ligand and receptorfamilies, however, homology identified between family members has beenfound mainly in the extracellular domain (“ECD”). Several of the TNFfamily cytokines, including TNF-α, Apo-1 ligand and CD40 ligand, arecleaved proteolytically at the cell surface; the resulting protein ineach case typically forms a homotrimeric molecule that functions as asoluble cytokine. TNF receptor family proteins are also usually cleavedproteolytically to release soluble receptor ECDs that can function asinhibitors of the cognate cytokines.

Recently, other members of the TNFR family have been identified. Suchnewly identified members of the TNFR family include CAR1, HVEM andosteoprotegerin (OPG) [Brojatsch et al., Cell, 87:845-855 (1996);Montgomery et al., Cell, 87:427-436 (1996); Marsters et al., J. Biol.Chem., 272:14029-14032 (1997); Simonet et al., Cell, 89:309-319 (1997)].Unlike other known TNFR-like molecules, Simonet et al., supra, reportthat OPG contains no hydrophobic transmembrane-spanning sequence.

For a review of the TNF family of cytokines and their receptors, seeGruss and Dower, supra.

Applicants herein describe the identification and characterization ofnovel polypeptides having homology to members of the tumor necrosisfactor family of polypeptides, designated herein as PRO715 polypeptides.

13. PRO353

The complement proteins comprise a large group of serum proteins some ofwhich act in an enzymatic cascade, producing effector molecules involvedin inflammation. The complement proteins are of particular importance inregulating movement and function of cells involved in inflammation.Given the physiological importance of inflammation and relatedmechanisms in vivo, efforts are currently being under taken to identifynew, native proteins which are involved in inflamation. We describeherein the identification and characterization of novel polypeptideswhich have homology to complement proteins, designated herein as PRO353polypeptides.

14. PRO361

The mucins comprise a family of glycoproteins which have been implicatedin carcinogenesis. Mucin and mucin-like proteins are secreted by bothnormal and transformed cells. Both qualitative and quantitative changesin mucins have been implicated in various types of cancer. Given themedical importance of cancer, efforts are currently being under taken toidentify new, native proteins which may be useful for the diagnosis ortreatment of cancer.

The chitinase proteins comprise a family of which have been implicatedin pathogenesis responses in plants. Chitinase proteins are produced byplants and microorganisms and may play a role in the defense of plantsto injury. Given the importance of plant defense mechanisms, efforts arecurrently being under taken to identify new, native proteins which maybe useful for modulation of pathogenesis-related responses in plants. Wedescribe herein the identification and characterization of novelpolypeptides which have homology to mucin and chitinase, designated inthe present application as PRO361 polypeptides.

15. PRO365

Polypeptides such as human 2-19 protein may function as cytokines.Cytokines are low molecular weight proteins which function to stimulateor inhibit the differentiation, proliferation or function of immunecells. Cytokines often act as intercellular messengers and have multiplephysiological effects. Given the physiological importance of immunemechanisms in vivo, efforts are currently being under taken to identifynew, native proteins which are involved in effecting the immune system.We describe herein the identification and characterization of novelpolypeptides which have homology to the human 2-19 protein, designatedheein as PRO365 polypeptides.

SUMMARY OF THE INVENTION

1. PRO241

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to biglycan protein, wherein the polypeptide isdesignated in the present application as “PRO241”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO241 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO241 polypeptidehaving amino acid residues 1 to 379 of FIG. 2 (SEQ ID NO:2), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO241polypeptide. In particular, the invention provides isolated nativesequence PRO241 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 379 of FIG. 2 (SEQ ID NO:2).Another embodiment of the present invention is directed to a PRO241polypeptide lacking the N-terminal signal peptide, wherein the PRO241polypeptide comprises about amino acids 16 to 379 of the full-lengthPRO241 amino acid sequence (SEQ ID NO: 2).

2. PRO243

Applicants have identified a cDNA clone (DNA35917-1207) that encodes anovel polypeptide, designated in the present application as “PRO243”.

In one embodiment, the invention provides an isolated nucleic acidmolecule having at least about 80% sequence identity to (a) a DNAmolecule encoding a PRO243 polypeptide comprising the sequence of aminoacids 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7), or (b) thecomplement of the DNA molecule of (a). The sequence identity preferablyis about 85%, more preferably about 90%, most preferably about 95%. Inone aspect, the isolated nucleic acid has at least about 80%, preferablyat least about 85%, more preferably at least about 90%, and mostpreferably at least about 95% sequence identity with a polypeptidehaving amino acid residues 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7).Preferably, the highest degree of sequence identity occurs within thefour (4) conserved cysteine clusters (amino acids 51 to 125; amino acids705 to 761; amino acids 784 to 849; and amino acids 897 to 931) of FIG.4 (SEQ ID NO:7). In a further embodiment, the isolated nucleic acidmolecule comprises DNA encoding a PRO243 polypeptide having amino acidresidues 1 or about 24 to 954 of FIG. 4 (SEQ ID NO:7), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the invention provides anucleic acid of the full length protein of clone DNA35917-1207,deposited with the ATCC under accession number ATCC 209508,alternatively the coding sequence of clone DNA35917-1207, depositedunder accession number ATCC 209508.

In yet another embodiment, the invention provides isolated PRO243polypeptide. In particular, the invention provides isolated nativesequence PRO243 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 or about 24 to 954 of FIG. 4 (SEQ IDNO:7). Native PRO243 polypeptides with or without the native signalsequence (amino acids 1 to 23 in FIG. 4 (SEQ ID NO:7)), and with orwithout the initiating methionine are specifically included.Alternatively, the invention provides a PRO243 polypeptide encoded bythe nucleic acid deposited under accession number ATCC 209508.

3. PRO299

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO299”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO299 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO299 polypeptidehaving amino acid residues 1 to 737 of FIG. 6 (SEQ ID NO:15), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO299polypeptide. In particular, the invention provides isolated nativesequence PRO299 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 737 of FIG. 6 (SEQ ID NO:15). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO299 polypeptide.

4. PRO323

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to a microsomal dipeptidase protein, wherein thepolypeptide is designated in the present application as “PRO323”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO323 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO323 polypeptidehaving amino acid residues 1 to 433 of FIG. 10 (SEQ ID NO:24), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO323polypeptide. In particular, the invention provides isolated nativesequence PRO323 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 433 of FIG. 10 (SEQ ID NO:24).

5. PRO327

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to prolactin receptor, wherein the polypeptide isdesignated in the present application as “PRO327”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO327 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO327 polypeptidehaving amino acid residues 1 to 422 of FIG. 14 (SEQ ID NO:32), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO327polypeptide. In particular, the invention provides isolated nativesequence PRO327 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 422 of FIG. 14 (SEQ ID NO:32).

6. PRO233

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO233”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO233 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO233 polypeptidehaving amino acid residues 1 to 300 of FIG. 16 (SEQ ID NO:37), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO233polypeptide. In particular, the invention provides isolated nativesequence PRO233 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 300 of FIG. 16 (SEQ ID NO:37).

7. PRO344

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptides are designated in the presentapplication as “PRO344”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO344 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO344 polypeptidehaving amino acid residues 1 to 243 of FIG. 18 (SEQ ID NO:42), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO344polypeptide. In particular, the invention provides isolated nativesequence PRO344 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 243 of FIG. 18 (SEQ ID NO:42).

8. PRO347

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to cysteine-rich secretory protein-3, wherein thepolypeptide is designated in the present application as “PRO347”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO347 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO347 polypeptidehaving amino acid residues 1 to 455 of FIG. 20 (SEQ ID NO:50), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO347polypeptide. In particular, the invention provides isolated nativesequence PRO347 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 455 of FIG. 20 (SEQ ID NO:50).

9. PRO354

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to the heavy chain of the inter-alpha-trypsin inhibitor(ITI), wherein the polypeptide is designated in the present applicationas “PRO354”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO354 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO354 polypeptidehaving amino acid residues 1 to 694 of FIG. 22 (SEQ ID NO:55), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO354polypeptide. In particular, the invention provides isolated nativesequence PRO354 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 694 of FIG. 22 (SEQ ID NO:55).

10. PRO355

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO355”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO355 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO355 polypeptidehaving amino acid residues 1 to 440 of FIG. 24 (SEQ ID NO:61), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO355polypeptide. In particular, the invention provides isolated nativesequence PRO355 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 440 of FIG. 24 (SEQ ID NO:61). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO355 polypeptide.

11. PRO357

Applicants have identified a cDNA clone that encodes a novel polypeptidehaving homology to insulin-like growth factor (IGF) acid labile subunit(ALS), wherein the polypeptide is designated in the present applicationas “PRO357”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO357 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO357 polypeptidehaving amino acid residues 1 through 598 of FIG. 26 (SEQ ID NO:69), oris complementary to such encoding nucleic acid sequence, and remainsstably bound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO357polypeptide. In particular, the invention provides isolated nativesequence PRO357 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 through 598 of FIG. 26 (SEQ IDNO:69). An additional embodiment of the present invention is directed toan isolated extracellular domain of a PRO357 polypeptide.

12. PRO715

Applicants have identified cDNA clones that encode novel polypeptideshaving homology to tumor necrosis factor family polypeptides, whereinthe polypeptides are designated in the present application as “PRO715”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO715 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO715 polypeptidehaving amino acid residues 1 to 250 of FIG. 28 (SEQ ID NO:76), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides isolated PRO715polypeptide. In particular, the invention provides isolated nativesequence PRO715 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 250 of FIG. 28 (SEQ ID NO:76). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO715 polypeptide.

13. PRO353

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptides are designated in the presentapplication as “PRO353”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO353 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO353 polypeptidehaving amino acid residues 1 to 281 of FIG. 30 (SEQ ID NO:78), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions.

In another embodiment, the invention provides an isolated PRO353polypeptide. In particular, the invention provides isolated nativesequence PRO353 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 281 of FIG. 30 (SEQ ID NO:78).

14. PRO361

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO361”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO361 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO361 polypeptidehaving amino acid residues 1 to 431 of FIG. 32 (SEQ ID NO:83), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. The isolated nucleic acid sequence may comprisethe cDNA insert of the vector deposited on Feb. 5, 1998 as ATCC 209621which includes the nucleotide sequence encoding PRO361.

In another embodiment, the invention provides isolated PRO361polypeptide. In particular, the invention provides isolated nativesequence PRO361 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 431 of FIG. 32 (SEQ ID NO:83). Anadditional embodiment of the present invention is directed to anisolated extracellular domain of a PRO361 polypeptide having amino acids1 to 379 of the amino acids sequence shown in FIG. 32 (SEQ ID NO:83).Optionally, the PRO361 polypeptide is obtained or is obtainable byexpressing the polypeptide encoded by the cDNA insert of the vectordeposited on Feb. 5, 1998 as ATCC 209621.

15. PRO365

Applicants have identified a cDNA clone that encodes a novelpolypeptide, wherein the polypeptide is designated in the presentapplication as “PRO365”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO365 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO365 polypeptidehaving amino acid residues 1 to 235 of FIG. 34 (SEQ ID NO:91), or iscomplementary to such encoding nucleic acid sequence, and remains stablybound to it under at least moderate, and optionally, under highstringency conditions. In another aspect, the isolated nucleic acidcomprises DNA encoding the PRO365 polypeptide having amino acid residues21 to 235 of FIG. 34 (SEQ ID NO:91), or is complementary to suchencoding nucleic acid sequence, and remains stably bound to it under atleast moderate, and optionally, under high stringency conditions.

In another embodiment, the invention provides isolated PRO365polypeptide. In particular, the invention provides isolated nativesequence PRO365 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 235 of FIG. 34 (SEQ ID NO:91). Anadditional embodiment of the present invention is directed to an aminoacid sequence comprising residues 21 to 235 of FIG. 34 (SEQ ID NO: 91).

16. Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein or the coding sequence of anyother specifically defined fragment of the full-length amino acidsequence as disclosed herein, or (b) the complement of the DNA moleculeof (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC as disclosed herein, or (b)the complement of the DNA molecule of (a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes, for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody or as antisense oligonucleotide probes. Such nucleicacid fragments are usually at least about 20 nucleotides in length,preferably at least about 30 nucleotides in length, more preferably atleast about 40 nucleotides in length, yet more preferably at least about50 nucleotides in length, yet more preferably at least about 60nucleotides in length, yet more preferably at least about 70 nucleotidesin length, yet more preferably at least about 80 nucleotides in length,yet more preferably at least about 90 nucleotides in length, yet morepreferably at least about 100 nucleotides in length, yet more preferablyat least about 110 nucleotides in length, yet more preferably at leastabout 120 nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a fall-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,preferably at least about 81% positives, more preferably at least about82% positives, yet more preferably at least about 83% positives, yetmore preferably at least about 84% positives, yet more preferably atleast about 85% positives, yet more preferably at least about 86%positives, yet more preferably at least about 87% positives, yet morepreferably at least about 88% positives, yet more preferably at leastabout 89% positives, yet more preferably at least about 90% positives,yet more preferably at least about 91% positives, yet more preferably atleast about 92% positives, yet more preferably at least about 93%positives, yet more preferably at least about 94% positives, yet morepreferably at least about 95% positives, yet more preferably at leastabout 96% positives, yet more preferably at least about 97% positives,yet more preferably at least about 98% positives and yet more preferablyat least about 99% positives when compared with the amino acid sequenceof a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO241 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA34392-1170”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO: 1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a native sequencePRO243 cDNA, wherein SEQ ID NO:6 is a clone designated herein as“DNA35917-1207”.

FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived from thecoding sequence of SEQ ID NO:6 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:14) of a native sequencePRO299 cDNA, wherein SEQ ID NO:14 is a clone designated herein as“DNA39976-1215”.

FIG. 6 shows the amino acid sequence (SEQ ID NO:15) derived from thecoding sequence of SEQ ID NO:14 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence designated herein as DNA28847 (SEQ IDNO:18).

FIG. 8 shows a nucleotide sequence designated herein as DNA35877 (SEQ IDNO:19).

FIG. 9 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO323 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA35595-1228”.

FIG. 10 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 9.

FIG. 11 shows a single-stranded nucleotide sequence (SEQ ID NO:29)containing the nucleotide sequence (nucleotides 79-1416) of a chimericfusion protein between a PRO323-derived polypeptide and a portion of anIgG constant domain, wherein the chimeric fusion protein is designatedherein as “PRO454”. The single-stranded nucleotide sequence (SEQ IDNO:29) encoding the PRO323/IgG fusion protein (PRO454) is designatedherein as “DNA35872”.

FIG. 12 shows the amino acid sequence (SEQ ID NO:30) derived fromnucleotides 79-1416 of the nucleotide sequence shown in FIG. 11. Thejunction in the PRO454 amino acid sequence between the PRO323-derivedsequences and the IgG-derived sequences appears between amino acids415-416 in the figure.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:31) of a native sequencePRO327 cDNA, wherein SEQ ID NO:31 is a clone designated herein as“DNA38113-1230”.

FIG. 14 shows the amino acid sequence (SEQ ID NO:32) derived from thecoding sequence of SEQ ID NO:31 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:36) of a native sequencePRO233 cDNA, wherein SEQ ID NO:36 is a clone designated herein as“DNA34436-1238”.

FIG. 16 shows the amino acid sequence (SEQ ID NO:37) derived from thecoding sequence of SEQ ID NO:36 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:41) of a native sequencePRO344 cDNA, wherein SEQ ID NO:41 is a clone designated herein as“DNA40592-1242”.

FIG. 18 shows the amino acid sequence (SEQ ID NO:42) derived from thecoding sequence of SEQ ID NO:41 shown in FIG. 17.

FIG. 19 shows a nucleotide sequence (SEQ ID NO:49) of a native sequencePRO347 cDNA, wherein SEQ ID NO:49 is a clone designated herein as“DNA44176-1244”.

FIG. 20 shows the amino acid sequence (SEQ ID NO:50) derived from thecoding sequence of SEQ ID NO:49 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:54) of a native sequencePRO354 cDNA, wherein SEQ ID NO:54 is a clone designated herein as“DNA44192-1246”.

FIG. 22 shows the amino acid sequence (SEQ ID NO:55) derived from thecoding sequence of SEQ ID NO:54 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:60) of a native sequencePRO355 cDNA, wherein SEQ ID NO:60 is a clone designated herein as“DNA39518-1247”.

FIG. 24 shows the amino acid sequence (SEQ ID NO:61) derived from thecoding sequence of SEQ ID NO:60 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:68) of a native sequencePRO357 cDNA, wherein SEQ ID NO:68 is a clone designated herein as“DNA44804-1248”.

FIG. 26 shows the amino acid sequence (SEQ ID NO:69) derived from thecoding sequence of SEQ ID NO:68 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:75) of a native sequencePRO715 cDNA, wherein SEQ ID NO:75 is a clone designated herein as“DNA52722-1229”.

FIG. 28 shows the amino acid sequence (SEQ ID NO:76) derived from thecoding sequence of SEQ ID NO:75 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:77) of a native sequencePRO353 cDNA, wherein SEQ ID NO:77 is a clone designated herein as“DNA41234-1242”.

FIG. 30 shows the amino acid sequence (SEQ ID NO:78) derived from thecoding sequence of SEQ ID NO:77 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:82) of a native sequencePRO361 cDNA, wherein SEQ ID NO:82 is a clone designated herein as“DNA45410-1250”.

FIG. 32 shows the amino acid sequence (SEQ ID NO:83) derived from thecoding sequence of SEQ ID NO:82 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:90) of a native sequencePRO365 cDNA, wherein SEQ ID NO:90 is a clone designated herein as“DNA46777-1253”.

FIG. 34 shows the amino acid sequence (SEQ ID NO:91) derived from thecoding sequence of SEQ ID NO:90 shown in FIG. 33.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are contemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

“PRO variant polynucleotide” or “PRO variant nucleic acid sequence”means a nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, more preferably at least about 81% nucleic acidsequence identity, more preferably at least about 82% nucleic acidsequence identity, more preferably at least about 83% nucleic acidsequence identity, more preferably at least about 84% nucleic acidsequence identity, more preferably at least about 85% nucleic acidsequence identity, more preferably at least about 86% nucleic acidsequence identity, more preferably at least about 87% nucleic acidsequence identity, more preferably at least about 88% nucleic acidsequence identity, more preferably at least about 89% nucleic acidsequence identity, more preferably at least about 90% nucleic acidsequence identity, more preferably at least about 91% nucleic acidsequence identity, more preferably at least about 92% nucleic acidsequence identity, more preferably at least about 93% nucleic acidsequence identity, more preferably at least about 94% nucleic acidsequence identity, more preferably at least about 95% nucleic acidsequence identity, more preferably at least about 96% nucleic acidsequence identity, more preferably at least about 97% nucleic acidsequence identity, more preferably at least about 98% nucleic acidsequence identity and yet more preferably at least about 99% nucleicacid sequence identity with a nucleic acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

 100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:

100 times the fraction W/Z

where W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions, see Table 6 below). For purposes herein, the% value of positives is determined by dividing (a) the number of aminoacid residues scoring a positive value between the PRO polypeptide aminoacid sequence of interest having a sequence derived from the native PROpolypeptide sequence and the comparison amino acid sequence of interest(i.e., the amino acid sequence against which the PRO polypeptidesequence is being compared) as determined in the BLOSUM62 matrix ofWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest.

Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:

100 times the fraction X/Y

where X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2in that program's alignment of A and B, and where Y is the total numberof amino acid residues in B. It will be appreciated that where thelength of amino acid sequence A is not equal to the length of amino acidsequence B, the % positives of A to B will not equal the % positives ofB to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein

% amino acid sequence identity=

(the number of identically matching amino acid residues between the twopolypeptide sequences as determined by ALIGN-2) divided by (the totalnumber of amino acid residues of the PRO polypeptide)=

5 divided by 15=33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein

% amino acid sequence identity=

(the number of identically matching amino acid residues between the twopolypeptide sequences as determined by ALIGN-2) divided by (the totalnumber of amino acid residues of the PRO polypeptide)=

5 divided by 10=50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA

% nucleic acid sequence identity=

(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence)=

6 divided by 14=42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides)

% nucleic acid sequence identity=

(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence)=

4 divided by 12=33.3%

II. Compositions and Methods of the Invention

A. Full-Length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

1. Full-length PRO241 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO241. In particular, Applicants have identified and isolated cDNAencoding a PRO241 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO241 polypeptide havecertain homology with the various biglycan proteins. Accordingly, it ispresently believed that PRO241 polypeptide disclosed in the presentapplication is a newly identified biglycan homolog polypeptide and maypossess activity typical of biglycan proteins.

2. Full-length PRO243 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO243. In particular, Applicants have identified and isolated cDNAencoding a PRO243 polypeptide, as disclosed in further detail in theExamples below. Using BLAST, BLAST-2 and FastA sequence alignmentcomputer programs, Applicants found that a full-length native sequencePRO243 (shown in FIG. 4 and SEQ ID NO:7) has certain amino acid sequenceidentity with African clawed frog and Xenopus chordin and certainhomology with rat chordin. Accordingly, it is presently believed thatPRO243 disclosed in the present application is a newly identified memberof the chordin protein family and may possess ability to influencenotochord and muscle formation by the dorsalization of the mesoderm.

3. Full-length PRO299

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO299. In particular, Applicants have identified and isolated cDNAencoding a PRO299 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO299polypeptide have certain homology with the notch protein. Accordingly,it is presently believed that PRO299 polypeptide disclosed in thepresent application is a newly identified member of the notch proteinfamily and possesses signaling properties typical of the notch proteinfamily.

4. Full-length PRO323 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO323. In particular, Applicants have identified and isolated cDNAencoding a PRO323 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO323polypeptide have certain homology with various dipeptidase proteins.Accordingly, it is presently believed that PRO323 polypeptide disclosedin the present application is a newly identified dipeptidase homologthat has dipeptidase activity.

5. Full-length PRO327 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO327. In particular, Applicants have identified and isolated cDNAencoding a PRO327 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO327 polypeptide havecertain homology with various prolactin receptor proteins. Accordingly,it is presently believed that PRO327 polypeptide disclosed in thepresent application is a newly identified prolactin receptor homolog andhas activity typical of a prolactin receptor protein.

6. Full-length PRO233 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO233. In particular, Applicants have identified and isolated cDNAencoding a PRO233 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO233polypeptide have certain homology with various reductase proteins.Applicants have also found that the DNA encoding the PRO233 polypeptidehas significant homology with proteins from Caenorhabditis elegans.Accordingly, it is presently believed that PRO233 polypeptide disclosedin the present application is a newly identified member of the reductasefamily and possesses the ability to effect the redox state of a celltypical of the reductase family.

7. Full-length PRO344 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO344. In particular, Applicants have identified and isolated cDNAencoding PRO344 polypeptides, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO344polypeptide have certain homology with the human and mouse complementproteins. Accordingly, it is presently believed that the PRO344polypeptide disclosed in the present application is a newly identifiedmember of the complement family and possesses the ability to affect theinflammation process as is typical of the complement family of proteins.

8. Full-length PRO347 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO347. In particular, Applicants have identified and isolated cDNAencoding a PRO347 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO347 polypeptide havecertain homology with various cysteine-rich secretory proteins.Accordingly, it is presently believed that PRO347 polypeptide disclosedin the present application is a newly identified cysteine-rich secretoryprotein and may possess activity typical of the cysteine-rich secretoryprotein family.

9. Full-length PRO354 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO354. In particular, Applicants have identified and isolated cDNAencoding a PRO354 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that portions of the PRO354 polypeptide havecertain homology with the inter-alpha-trypsin inhibitor heavy chainprotein. Accordingly, it is presently believed that PRO354 polypeptidedisclosed in the present application is a newly identifiedinter-alpha-trypsin inhibitor heavy chain homolog.

10. Full-length PRO355 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO355. In particular, Applicants have identified and isolated cDNAencoding a PRO355 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO355polypeptide have certain homology with the CRTAM protein. Applicantshave also found that the DNA encoding the PRO355 polypeptide also hashomology to the thymocyte activation and developmental protein, the H20Areceptor, the H20B receptor, the poliovirus receptor and theCercopithecus aethiops AGM delta 1 protein. Accordingly, it is presentlybelieved that PRO355 polypeptide disclosed in the present application isa newly identified member of the CRTAM protein family.

11. Full-length PRO357 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO357. In particular, Applicants have identified and isolated cDNAencoding a PRO357 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO357polypeptide have certain homology with the acid labile subunit ofinsulin-like growth factor. Applicants have also found that non-codingregions of the DNA44804-1248 align with a human gene signature asdescribed in WO 95/14772. Applicants have further found that non-codingregions of the DNA44804-1248 align with the adenovirus type 12/humanrecombinant viral DNA as described in Deuring and Doerfler, Gene,26:283-289 (1983) Based on the coding region homology, it is presentlybelieved that PRO357 polypeptide disclosed in the present application isa newly identified member of the leucine rich repeat family of proteins,and particularly, is related to the acid labile subunit of insulin-likegrowth factor. As such, PRO357 is likely to be involved in bindingmechanisms, and may be part of a complex.

12. Full-length PRO715 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO715. In particular, Applicants have identified and isolated cDNAmolecules encoding PRO715 polypeptides, as disclosed in further detailin the Examples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO715polypeptides have certain homology with the various members of the tumornecrosis family of proteins. Accordingly, it is presently believed thatthe PRO715 polypeptides disclosed in the present application are newlyidentified members of the tumor necrosis factor family of proteins.

13. Full-length PRO353 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO353. In particular, Applicants have identified and isolated cDNAencoding PRO353 polypeptides, as disclosed in further detail in theExamples below. Using BLAST and, FastA sequence alignment computerprograms, Applicants found that various portions of the PRO353polypeptides have certain homology with the human and mouse complementproteins. Accordingly, it is presently believed that the PRO353polypeptides disclosed in the present application are newly identifiedmembers of the complement protein family and possesses the ability toeffect the inflammation process as is typical of the complement familyof proteins.

14. Full-length PRO361 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO361. In particular, Applicants have identified and isolated cDNAencoding a PRO361 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO361polypeptide have certain homology with the mucin and chitinase proteins.Accordingly, it is presently believed that PRO361 polypeptide disclosedin the present application is a newly identified member of the mucinand/or chitinase protein families and may be associated with cancer,plant pathogenesis or receptor functions typical of the mucin andchitinase protein families, respectively.

15. Full-length PRO365 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO365. In particular, Applicants have identified and isolated cDNAencoding a PRO365 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that various portions of the PRO365polypeptide have certain homology with the human 2-19 protein.Accordingly, it is presently believed that PRO365 polypeptide disclosedin the present application is a newly identified member of the human2-19 protein family.

2. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No.5,364,934. Variationsmay be a substitution, deletion or insertion of one or more codonsencoding the PRO that results in a change in the amino acid sequence ofthe PRO as compared with the native sequence PRO. Optionally thevariation is by substitution of at least one amino acid with any otheramino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 1 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; ieu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mut agenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 53 317:415 (1986)] or other known techniques can beperformed on the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol. 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC- terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep.11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)1; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis W. H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology 185:527-537 (1990) and Mansour et al., Nature336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e-g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published May 2, 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., BiolTechnology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida, Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J. 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinesehamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-tppe DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphateisomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used for tissue typing, wherein the PRO polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another. PRO nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μmg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1lacZ reportergene under control of a GAL1-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix-see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al.,Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense-Okano, Neurochem., 56:560(1991); Olipodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology 4:469-471 (1994), and PCT publication No. WO 97/33551 (publishedSep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO go 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

PRO241 polypeptides of the present invention which possess biologicalactivity related to that of the endogenous biglycan protein may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO241polypeptides of the present invention for such purposes.

Chordin is a candidate gene for a dysmorphia syndrome known as Corneliade Lange Syndrome (CDL) which is characterized by distinctive facialfeatures (low anterior hairline, synophrys, antenerted nares, maxillaryprognathism, long philtrum, ‘carp’ mouth), prenatal and postnatal growthretardation, mental retardation and, often but not always, upper limbabnormalities. There are also rare cases where CDL is present inassociation with thrombocytopenia. The gene for CDL has been mapped bylinkage to 3q26.3 (OMIM #122470). Xchd involvement in early Xenopuspatterning and nervous system development makes CHD in intriguingcandidate gene. CHD maps to the appropriate region on chromosome 3. Itis very close to THPO, and deletions encompassing both THPO and CHDcould result in rare cases of thrombocytopenia and developmentalabnormalities. In situ analysis of CD revealed that almost all adulttissues are negative for CHD expression, the only positive signal wasobserved in the cleavage line of the developing synovial joint formingbetween the femoral head and acetabulum (hip joint) implicating CHD inthe development and presumably growth of long bones. Such a function, ifdisrupted, could result in growth retardation.

The human CHD amino acid sequence predicted from the cDNA is 50%identical (and 66% conserved) to Xchd. All 40 cysteines in the 4cysteine-rich domains are conserved. These cysteine rich domains aresimilar to those observed in thrombospondin, procollagen and vonWillebrand factor. Bornstein, P. FASEB J 6: 3290-3299 (1992); Hunt, L. &Barker, W. Biochem. Biophys. Res. Commun. 144: 876-882 (1987).

The human CHD locus (genomic PRO243) comprises 23 exons in 9.6 kb ofgenomic DNA. The initiating methionine is in exon 1 and the stop codonin exon 23. A CpG island is located at the 5′ and of the gene, beginningapproximately 100 bp 5′ of exon 1 and extends through the first exon andends within the first intron. The THPO and CHD loci are organized in ahead-to-head fashion with approximately 2.2 kb separating theirtranscription start sites. At the protein level, PRO243 is 51% identicalto Xenopus chordin (Xchd). All forty cysteines in the one amino terminaland three carboxy terminal cysteine-rich clusters are conserved.

PRO243 is a 954 amino acid polypeptide having a signal sequence atresidues 1 to about 23. There are 4 cysteine clusters: (1) residuesabout 51 to about 125; (2) residues about 705 to about 761; (3) residuesabout 784 to about 849; and (4) residues about 897 to about 931. Thereare potential leucine zippers at residues about 315 to about 396, andN-glycosylation sites at residues 217, 351, 365 and 434.

PRO299 polypeptides and portions thereof which have homology to thenotch protein may be useful for in vivo therapeutic purposes, as well asfor various other applications. The identification of novel notchproteins and related molecules may be relevant to a number of humandisorders such as those effecting development. Thus, the identificationof new notch proteins and notch-like molecules is of special importancein that such proteins may serve as potential therapeutics for a varietyof different human disorders. Such polypeptides may also play importantroles in biotechnological and medical research as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO299.

PRO323 polypeptides of the present invention which possess biologicalactivity related to that of one or more endogenous dipeptidase proteinsmay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO323 polypeptides of the present invention for such purposes.

PRO327 polypeptides of the present invention which possess biologicalactivity related to that of the endogenous prolactin receptor proteinmay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO327 polypeptides of the present invention for such purposes. PRO327polypeptides which possess the ability to bind to prolactin may functionboth in vitro and in vivo as prolactin antagonists.

PRO233 polypeptides and portions thereof which have homology toreductase may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novel reductaseproteins and related molecules may be relevant to a number of humandisorders such as inflammatory disease, organ failure, atherosclerosis,cardiac injury, infertility, birth defects, premature aging, AIDS,cancer, diabetic complications and mutations in general. Given thatoxygen free radicals and antioxidants appear to play important roles ina number of disease processes, the identification of new reductaseproteins and reductase-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO233.

PRO344 polypeptides and portions thereof which have homology tocomplement proteins may also be useful for in vivo therapeutic purposes,as well as for various other applications. The identification of novelcomplement proteins and related molecules may be relevant to a number ofhuman disorders such as effecting the inflammatory response of cells ofthe immune system. Thus, the identification of new complement proteinsand complement-like molecules is of special importance in that suchproteins may serve as potential therapeutics for a variety of differenthuman disorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO344.

PRO347 polypeptides of the present invention which possess biologicalactivity related to that of cysteine-rich secretory proteins may beemployed both in vivo for therapeutic purposes and in vitro. Those ofordinary skill in the art will well know how to employ the PRO347polypeptides of the present invention for such purposes.

PRO354 polypeptides of the present invention which possess biologicalactivity related to that of the heavy chain of the inter-alpha-trypsininhibitor protein may be employed both in vivo for therapeutic purposesand in vitro. Those of ordinary skill in the art will well know how toemploy the PRO354 polypeptides of the present invention for suchpurposes.

PRO355 polypeptides and portions thereof which have homology to CRTAMmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel moleculesassociated with T cells may be relevant to a number of human disorderssuch as conditions involving the immune system in general. Given thatthe CRTAM protein binds antibodies which play important roles in anumber of disease processes, the identification of new CRTAM proteinsand CRTAM-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research, as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO355.

PRO357 can be used in competitive binding assays with ALS to determineits activity with respect to ALS. Moreover, PRO357 can be used in assaysto determine if it prolongs polypeptides which it may complex with tohave longer half-lives in vivo. PRO357 can be used similarly in assayswith carboxypeptidase, to which it also has homology. The results can beapplied accordingly.

PRO715 polypeptides of the present invention which possess biologicalactivity related to that of the tumor necrosis factor family of proteinsmay be employed both in vivo for therapeutic purposes and in vitro.Those of ordinary skill in the art will well know how to employ thePRO715 polypeptides of the present invention for such purposes. PRO715polypeptides will be expected to bind to their specific receptors,thereby activating such receptors. Variants of the PRO715 polypeptidesof the present invention may function as agonists or antagonists oftheir specific receptor activity.

PRO353 polypeptides and portions thereof which have homology to thecomplement protein may also be useful for in vivo therapeutic purposes,as well as for various other applications. The identification of novelcomplement proteins and related molecules may be relevant to a number ofhuman disorders such as effecting the inflammatory response of cells ofthe immune system. Thus, the identification of new complement proteinscomplement-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO353.

PRO361 polypeptides and portions thereof which have homology to mucinand/or chitinase proteins may also be useful for in vivo therapeuticpurposes, as well as for various other applications. The identificationof novel mucin and/or chitinase proteins and related molecules may berelevant to a number of human disorders such as cancer or thoseinvolving cell surface molecules or receptors. Thus, the identificationof new mucin and/or chitinase proteins is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO361.

PRO365 polypeptides and portions thereof which have homology to thehuman 2-19 protein may also be useful for in vivo therapeutic purposes,as well as for various other applications. The identification of novelhuman 2-19 proteins and related molecules may be relevant to a number ofhuman disorders such as modulating the binding or activity of cells ofthe immune system. Thus, the identification of new human 2-19 proteinsand human 2-19 protein-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO365.

F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subcloned may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra ] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann etal., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].The techniques of Cole et al. and Boerner et al. are also available forthe preparation of human monoclonal antibodies (Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner etal., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies canbe made by introducing of human immunoglobulin loci into transgenicanimals, e.g., mice in which the endogenous immunoglobulin genes havebeen partially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, for example, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inthe following scientific publications: Marks et al., Bio/Technology 10,779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonbergand Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature 305:537-539 (1983)]. Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published May13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CHI) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217-225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD 16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148:2918-2922(1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, andPEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extrudedthrough filters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to beintermolecularS-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulthydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

G. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSephadex resin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing the PROto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1

Extracellular Domain Homology Screening to Identify Novel Polypeptidesand cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI sites.

Example 2

Isolation of cDNA Clones by Amylase Screening

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI Tinkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176-179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water.

The sequence of the forward oligonucleotide 1 was:

5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:16)

The sequence of reverse oligonucleotide 2 was:

5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO:17)

PCR was then performed as follows:

a. Denature 92° C.,  5 minutes b. 3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c. 3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3

Isolation of cDNA Clones Encoding Human PRO241

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA30876. Based on the DNA30876 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO241.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-GGAAATGAGTGCAAACCCTC-3′ (SEQ ID NO:3) reverse PCRprimer 5′-TCCCAAGCTGAACACTCATTCTGC-3′ (SEQ ID NO:4)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30876 sequence which had the followingnucleotide sequence

hybridization probe

5′-GGGTGACGGTGTTCCATATCAGAATTGCAGAAGCAAAACTGACCTCAGTT-3′ (SEQ ID NO:5)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO241 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB29).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO241 [herein designated as DNA34392-1170](SEQ ID NO:1) and the derived protein sequence for PRO241.

The entire nucleotide sequence of DNA34392-1170 is shown in FIG. 1 (SEQID NO:1). Clone DNA34392-1170 contains a single open reading frame withan apparent translational initiation site at nucleotide positions234-236 and ending at the stop codon at nucleotide positions 1371-1373(FIG. 1). The predicted polypeptide precursor is 379 amino acids long(FIG. 2). The full-length PRO241 protein shown in FIG. 2 has anestimated molecular weight of about 43,302 daltons and a pI of about7.30. Clone DNA34392-1170 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209526.

Analysis of the amino acid sequence of the full-length PRO241polypeptide suggests that it possess significant homology to the variousbiglycan proteoglycan proteins, thereby indicating that PRO241 is anovel biglycan homolog polypeptide.

Example 4

Isolation of cDNA Clones Encoding Human PRO243 by Genomic Walking

Introduction: Human thrombopoietin (THPO) is a glycosylated hormone of352 amino acids consisting of two domains. The N-terminal domain,sharing 50% similarity to erythropoietin, is responsible for thebiological activity. The C-terminal region is required for secretion.The gene for thrombopoietin (THPO) maps to human chromosome 3q27-q28where the six exons of this gene span 7 kilobase base pairs of genomicDNA (Gurney et al., Blood 85: 981-988 (1995). In order to determinewhether there were any genes encoding THPO homologues located in closeproximity to THPO, genomic DNA fragments from this region wereidentified and sequenced. Three P1 clones and one PAC clones (GenomeSystems Inc., St. Louis, Mo.; cat. Nos. P1-2535 and PAC-6539)encompassing the THPO locus were isolated and a 140 kb region wassequenced using the ordered shotgun strategy (Chen et al., Genomics 17:651-656 (1993)), coupled with a PCR-based gap filling approach. Analysisreveals that the region is gene-rich with four additional genes locatedvery close to THPO: tumor necrosis factor-receptor type 1 associatedprotein 2 (TRAP2) and elongation initiation factor gamma (elF4g),chloride channel 2 (CLCN2) and RNA polymerase II subunit hRPB17. Whileno THPO homolog was found in the region, four novel genes have beenpredicted by computer-assisted gene detection (GRAIL)(Xu et al., Gen.Engin. 16: 241-253 (1994), the presence of CpG islands (Cross, S. andBird, A., Curr. Opin. Genet. & Devel. 5: 109-314 (1995), and homology toknown genes (as detected by WU-BLAST2.0)(Altschul and Gish, MethodsEnzymol. 266: 460-480 (1996) (http://blast.wustl.edu/blast/README.html).

P1 and PAC clones: The initial human P1 clone was isolated from agenomic P1 library (Genome Systems Inc., St. Louis, Mo.; cat. no.:P1-2535) screened with PCR primers designed from the THPO genomicsequence (A. L. Gurney, et al., Blood 85: 981-88 (1995). PCR primerswere designed from the end sequences derived from this P1 clone werethen used to screen P1 and PAC libraries (Genome Systems, Cat. Nos.:P1-2535 & PAC-6539) to identify overlapping clones.

Ordered Shotgun Strategy: The Ordered Shotgun Strategy (OSS) (Chen etal., Genomics 17: 651-656 (1993)) involves the mapping and sequencing oflarge genomic DNA clones with a hierarchical approach. The P1 or PACclone was sonicated and the fragments subcloned into lambda vector(λBluestar) (Novagen, Inc., Madison, Wis.; cat. no. 69242-3). The lambdasubclone inserts were isolated by long-range PCR (Barnes, W. Proc. Natl.Acad. Sci. USA 91: 2216-2220 (1994) and the ends sequenced. Thelambda-end sequences were overlapped to create a partial map of theoriginal clone. Those lambda clones with overlapping end-sequences wereidentified, the insets subcloned into a plasmid vector (pUC9 or pUC18)and the ends of the plasmid subclones were sequenced and assembled togenerate a contiguous sequence. This directed sequencing strategyminimizes the redundancy required while allowing one to scan for andconcentrate on interesting regions.

In order to define better the THPO locus and to search for other genesrelated to the hematopoietin family, four genomic clones were isolatedfrom this region by PCR screening of human P1 and PAC libraries (GenomeSystem, Inc., Cat. Nos.: P1-2535 and PAC-6539). The sizes of the genomicfragments are as follows: P1.t is 40 kb; P1.g is 70 kb; P1.u is 70 kb;and PAC.z is 200 kb. Approximately 80% of the 200 kb genomic DNA regionwas sequenced by the Ordered Shotgun Strategy (OSS) (Chen et al.,Genomics 17: 651-56 (1993), and assembled into contigs usingAutoAssembler™ (Applied Biosystems, Perkin Elmer, Foster City, Calif.,cat. no. 903227). The preliminary order of these contigs was determinedby manual analysis. There were 46 contigs and filling in the gaps wasemployed. Table 7 summarized the number and sizes of the gaps.

TABLE 7 Summary of the gaps in the 140 kb region Size of gap number  <50bp 13  50-150 bp 7 150-300 bp 7  300-1000 bp 10 1000-5000 bp 7 >5000 bp2 (15,000 bp)

DNA sequencing: ABI DYE-primer™ chemistry (PE Applied Biosystems, FosterCity, Calif.; Cat. No.: 402112) was used to end-sequence the lambda andplasmid subclones. ABI DYE-terminater™ chemistry (PE Applied Biosystems,Foster City, Calif., Cat. No: 403044) was used to sequence the PCRproducts with their respective PCR primers. The sequences were collectedwith an AB1377 instrument. For PCR products larger than 1 kb, walkingprimers were used. The sequences of contigs generated by the OSSstrategy in AutoAssembler™ a (PE Applied Biosystems, Foster City,Calif.; Cat. No: 903227) and the gap-filling sequencing trace files wereimported into Sequencher™ (Gene Codes Corp., Ann Arbor, Mich.) foroverlapping and editing.

PCR-Based gapfilling Strategy: Primers were designed based on the 5′-and 3′-end sequenced of each contig, avoiding repetitive and low qualitysequence regions. All primers were designed to be 19-24-mers with 50-70%G/C content. Oligos were synthesized and gel-purified by standardmethods.

Since the orientation and order of the contigs were unknown,permutations of the primers were used in the amplification reactions.Two PCR kits were used: first, XL PCR kit (Perkin Elmer, Norwalk, Conn.;Cat. No.: N8080205), with extension times of approximately 10 minutes;and second, the Taq polymerase PCR kit (Qiagen Inc., Valencia, Calif.;Cat. No.: 201223) was used under high stringency conditions if smearedor multiple products were observed with the XL PCR kit. The main PCRproduct from each successful reactions was extracted from a 0.9% lowmelting agarose gel and purified with the Geneclean DNA Purification kitprior to sequencing.

Analysis: The identification and characterization of coding regions wascarried out as follows: First, repetitive sequences were masked usingRepeatMasker (A. F. A. Smit & P. Green,http://ftp.genome.washington.edu/RM/RM_details.html)which screens DNAsequences in FastA format against a library of repetitive elements andreturns a masked query sequence. Repeats not masked were identified bycomparing the sequence to the GenBank database using WUBLAST (Altschul,S & Gish, W., Methods Enzymol. 266: 460-480 (1996) and were maskedmanually.

Next, known genes were revealed by comparing the genomic regions againstGenentech's protein database using the WUBLAST2.0 algorithm and thenannotated by aligning the genomic and cDNA sequences for each gene,respectively, using a Needleman-Wunch (Needleman and Wunsch, J. Mol.Biol. 48: 443-453 (1970) algorithm to find regions of local identitybetween sequences which are otherwise largely dissimilar. The strategyresults in detection of all exons of the five known genes in the region,THPO, TRAP2, elF4g, CLCN2 and hRPB17 (Table 8).

TABLE 8 Summary of known genes located in the 140 kb region analyzedKnown genes Map position eukaryotic translation initiation factor 4gamma 3q27-qter thrombopoietin 3q26-q27 chloride channel 2 3q26-qter TNFreceptor associated protein 2 not previously mapped RNA polymerase IIsubunit hRPB17 not previously mapped

Finally, novel transcription units were predicted using a number ofapproaches. CpG islands (S. Cross & Bird, A., Curr. Opin. Genet. Dev. 5:109-314 (1995) islands were used to defme promoter regions and wereidentified as clusters of sites cleaved by enzymes recognizing GC-rich,6 or 8-mer palidromic sequences. CpG islands are usually associated withpromoter regions of genes. WUBLAST2.0 analysis of short genomic regions(10-20 kb) versus GenBank revealed matches to ESTs. The individual ESTsequences (or where possible, their sequence chromatogram files) wereretrieved and assembled with Sequencher to provide a theoretical cDNAsequence (designated herein as DNA34415). GRAIL2 (ApoCom Inc.,Knoxville, Tenn., command line version for the DEC alpha) was used topredict a novel exon. The five known genes in the region served asinternal controls for the success of the GRAIL algorithm.

Isolation: Chordin cDNA clones were isolated from an oligo-dT-primedhuman fetal lung library. Human fetal lung polyA⁺ RNA was purchased fromClontech (cat #6528-1, lot #43777) and 5 mg used to construct a cDNAlibrary in pKR5B (Genentech, LIB26). The 3′-primer(pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT) (SEQ ID NO:8) and the5′-linker (pCGGACGCGTGGGGCCTGCGCACCCAGCT) (SEQ ID NO:9) were designed tointroduce SalI and NotI restriction sites. Clones were screened witholigonucleotide probes designed from the putative human chordin cDNAsequence (DNA34415) deduced by manually “splicing” together the proposedgenomic exons of the gene. PCR primers flanking the probes were used toconfirm the identity of the cDNA clones prior to sequencing.

The screening oligonucleotides probes were the following:

OLI5640 34415.p1 5′-GCCGCTCCCCGAACGGGCAGCGGCTCCTTCTCAGAA-3′ (SEQ IDNO:10) and OLI5642 34415.p2 5′-GGCGCACAGCACGCAGCGCATCACCCCGAATGGCTC-3′(SEQ ID NO: 11); and

the flanking probes used were the following:

OLI5639 34415.f1 5′-GTGCTGCCCATCCGTTCTGAGAAGGA-3′ (SEQ ID NO:12) andOLI5643 34415.r 5′-GCAGGGTGCTCAAACAGGACAC-3′ (SEQ ID NO:13).

Example 5

Northern Blot and in situ RNA Hybridization Analysis of PRO243

Expression of PRO243 mRNA in human tissues was examined by Northern blotanalysis. Human polyA+ RNA blots derived from human fetal and adulttissues (Clontech, Palo Alto, Calif.; Cat. Nos. 7760-1 and 7756-1) werehybridized to a ³²P-labelled cDNA fragments probe based on the fulllength PRO243 cDNA. Blots were incubated with the probes inhybridization buffer (5×SSPE; 2×Denhardt's solution; 100 mg/mL denaturedsheared salmon sperm DNA; 50% formamide; 2% SDS) for 60 hours at 42° C.The blots were washed several times in 2×SSC; 0.05% SDS for 1 hour atroom temperature, followed by a high stringency wash 30 minute wash in0.1×SSC; 0.1% SDS at 50° C. and autoradiographed. The blots weredeveloped after overnight exposure by phosphorimager analysis (Fuji).

PRO243 mRNA transcripts were detected. Analysis of the expressionpattern showed the strongest signal of the expected 4.0 kb transcript inadult and fetal liver and a very faint signal in the adult kidney. Fetalbrain, lung and kidney were negative, as were adult heart, brain, lungand pancreas. Smaller transcripts were observed in placenta (2.0 kb),adult skeletal muscle (1.8 kb) and fetal liver (2.0 kb).

In situ hybridization of adult human tissue of PRO243 gave a positivesignal in the cleavage line of the developing synovial joint formingbetween the femoral head and acetabulum. All other tissues werenegative. Additional sections of human fetal face, head, limbs and mouseembryos were examined. Expression in human fetal tissues was observedadjacent to developing limb and facial bones in the perosteal msenchyme.The expression was highly specific and was often adjacent to areasundergoing vascularization. Expression was also observed in thedeveloping temporal and occipital lobes of the fetal brain, but was notobserved elsewhere in the brain. In addition, expression was seen in theganglia of the developing inner ear. No expression was seen in any ofthe mouse tissues with the human probes.

In situ hybridization was performed using an optimized protocol, usingPCR-generating ³³P-labeled riboprobes. (Lu and Gillett, Cell Vision 1:169-176 (1994)). Formalin-fixed, paraffin-embedded human fetal and adulttissues were sectioned, deparaffinized, deproteinated in proteinase K(20 g/ml) for 15 minutes at 37° C., and further processed for in situhybridization as described by Lu and Gillett (1994). A [³³P]-UTP-labeledantisense riboprobe was generated from a PCR product and hybridized at55° C. overnight. The slides were dipped in Kodak NTB2 nuclear trackemulsion and exposed for 4 weeks.

Example 6

Isolation of cDNA Clones Encoding Human PRO299

A cDNA sequence designated herein as DNA28847 (FIG. 7; SEQ ID NO:18) wasisolated as described in Example 2 above. After further analysis, a 3′truncated version of DNA28847 was found and is herein designatedDNA35877 (FIG. 8; SEQ ID NO:19). Based on the DNA35877 sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO299. Forwardand reverse PCR primers generally range from 20 to 30 nucleotides andare often designed to give a PCR product of about 100-1000 bp in length.The probe sequences are typically 40-55 bp in length. In some cases,additional oligonucleotides are synthesized when the consensus sequenceis greater than about 1-1.5 kbp. In order to screen several librariesfor a full-length clone, DNA from the libraries was screened by PCRamplification, as per Ausubel et al., Current Protocols in MolecularBiology with the PCR primer pair. A positive library was then used toisolate clones encoding the gene of interest using the probeoligonucleotide and one of the primer pairs.

Forward and reverse PCR primers were synthesized:

forward PCR primer 5′-CTCTGGAAGGTCACGGCCACAGG-3′ (SEQ ID NO:20) reversePCR primer 5′-CTCAGTTCGGTTGGCAAAGCTCTC-3′ (SEQ ID NO:21)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA35877 sequence which had the followingnucleotide sequence

hybridization probe

5′-CAGTGCTCCCTCATAGATGGACGAAAGTGTGACCCCCCTTTCAGGCGAGAGCTTTGCCAACCG (SEQID NO:22) AACTGA-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO299 sequence using the probeoligonucleotide.

RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or PRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO299 [herein designated as DNA39976-1215](SEQ ID NO:14) and the derived protein sequence for PRO299.

The entire nucleotide sequence of DNA39976-1215 is shown in FIG. 5 (SEQID NO:14). Clone DNA39976-1215 contains a single open reading frame withan apparent translational initiation site at nucleotide positions111-113 and ending at the stop codon at nucleotide positions 2322-2324(FIG. 5). The predicted polypeptide precursor is 737 amino acids long(FIG. 6). Important regions of the polypeptide sequence encoded by cloneDNA39976-1215 have been identified and include the following: a signalpeptide corresponding to amino acids 1-28, a putative transmembraneregion corresponding to amino acids 638-662, 10 EGF repeats,corresponding to amino acids 80-106, 121-203, 336-360, 378-415, 416-441,454-490, 491-528, 529-548, 567-604, and 605-622, respectively, and 10potential N-glycosylation sites, corresponding to amino acids 107-120,204-207, 208-222, 223-285, 286-304, 361-374, 375-377, 442-453, 549-563,and 564-566, respectively. Clone DNA39976-1215 has been deposited withATCC and is assigned ATCC deposit no. ATCC 209524.

Analysis of the amino acid sequence of the full-length PRO299polypeptide suggests that portions of it possess significant homology tothe notch protein, thereby indicating that PRO299 may be a novel notchprotein homolog and have activity typical of the notch protein.

Example 7

Isolation of cDNA Clones Encoding Human PRO323

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA30875. Based on the DNA30875 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO323.

PCR primers (two forward and one reverse) were synthesized:

forward PCR primer 1 5′-AGTTCTGGTCAGCCTATGTGCC-3′ (SEQ ID NO:25) forwardPCR primer 2 5′-CGTGATGGTGTCTTTGTCCATGGG-3′ (SEQ ID NO:26) reverse PCRprimer 5′-CTCCACCAATCCCGATGAACTTGG-3′ (SEQ ID NO:27)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30875 sequence which had the followingnucleotide sequence

hybridization probe

5′-GAGCAGATTGACCTCATACGCCGCATGTGTGCCTCCTATTCTGAGCTGGA-3′ (SEQ ID NO:28)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO323 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal liver tissue (LIB6).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO323 [herein designated as DNA35595-1228](SEQ ID NO:23) and the derived protein sequence for PRO323.

The entire nucleotide sequence of DNA35595-1228 is shown in FIG. 9 (SEQID NO:23). Clone DNA35595-1228 contains a single open reading frame withan apparent translational initiation site at nucleotide positions110-112 and ending at the stop codon at nucleotide positions 1409-1411(FIG. 9). The predicted polypeptide precursor is 433 amino acids long(FIG. 10). The full-length PRO323 protein shown in FIG. 10 has anestimated molecular weight of about 47,787 daltons and a pI of about6.11. Clone DNA35595-1228 has been deposited with ATCC and is assignedATCC deposit no. 209528.

Analysis of the amino acid sequence of the full-length PRO323polypeptide suggests that portions of it possess significant homology tovarious dipeptidase proteins, thereby indicating that PRO323 may be anovel dipeptidase protein.

Example 8

Isolation of cDNA Clones Encoding Human PRO327

An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and various ESTsequences were identified which showed certain degrees of homology tohuman prolactin receptor protein. Those EST sequences were aligned usingphrap and a consensus sequence was obtained. This consensus DNA sequencewas then extended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above. The extended assembly sequence is herein designatedDNA38110. The above searches were performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

Based upon the DNA38110 consensus sequence obtained as described above,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO327.

PCR primers (forward and reverse) were synthesized as follows:

forward PCR primer 5′-CCCGCCCGACGTGCACGTGAGCC-3′ (SEQ ID NO:33) reversePCR primer 5′-TGAGCCAGCCCAGGAACTGCTTG-3′ (SEQ ID NO:34)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA38110 consensus sequence which had thefollowing nucleotide sequence

hybridization probe

5′-CAAGTGCGCTGCAACCCCTTTGGCATCTATGGCTCCAAGAAAGCCGGGAT-3′ (SEQ ID NO:35)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO327 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue (LIB26).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO327 [herein designated as DNA38113-1230](SEQ ID NO:16) and the derived protein sequence for PRO327.

The entire nucleotide sequence of DNA38113-1230 is shown in FIG. 13 (SEQID NO:31). Clone DNA38113-1230 contains a single open reading frame withan apparent translational initiation site at nucleotide positions119-121 and ending at the stop codon at nucleotide positions 1385-1387(FIG. 13). The predicted polypeptide precursor is 422 amino acids long(FIG. 14). The full-length PRO327 protein shown in FIG. 14 has anestimated molecular weight of about 46,302 daltons and a pI of about9.42. Clone DNA38113-1230 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209530.

Analysis of the amino acid sequence of the full-length PRO327polypeptide suggests that it possess significant homology to the humanprolactin receptor protein, thereby indicating that PRO327 may be anovel prolactin binding protein.

Example 9

Isolation of cDNA Clones Encoding Human PRO233

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA30945. Based on the DNA30945 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO233.

PCR primers were synthesized as followed:

forward PCR primer 5′-GGTGAAGGCAGAAATTGGAGATG-3′ (SEQ ID NO:38) reversePCR primer 5′-ATCCCATGCATCAGCCTGTTTACC-3′ (SEQ ID NO:39)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30945 sequence which had the followingnucleotide sequence

hybridization probe

5′-GCTGGTGTAGTCTATACATCAGATTTGTTTGCTACACAAGATCCTCAG-3′ (SEQ ID NO:40)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO233 gene using the probe oligonucleotide.RNA for construction of the cDNA libraries was isolated from human fetalbrain tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO233 [herein designated as DNA34436-1238](SEQ ID NO:36) and the derived protein sequence for PRO233.

The entire nucleotide sequence of DNA34436-1238 is shown in FIG. 15 (SEQID NO:36). Clone DNA34436-1238 contains a single open reading frame withan apparent translational initiation site at nucleotide positions101-103 and ending at the stop codon at nucleotide positions 1001-1003(FIG. 15). The predicted polypeptide precursor is 300 amino acids long(FIG. 16). The full-length PRO233 protein shown in FIG. 16 has anestimated molecular weight of about 32,964 daltons and a pI of about9.52. In addition, regions of interest including the signal peptide anda putative oxidoreductase active site, are designated in FIG. 16. CloneDNA34436-1238 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209523

Analysis of the amino acid sequence of the full-length PRO233polypeptide suggests that portions of it possess significant homology tovarious reductase proteins, thereby indicating that PRO233 may be anovel reductase.

Example 10

Isolation of cDNA Clones Encoding Human PRO344

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA34398. Based on the DNA34398 consensus sequencs,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO344.

Based on the DNA34398 consensus sequence, forward and reverse PCRprimers were synthesized as follows:

forward PCR primer (34398.f1) 5′-TACAGGCCCAGTCAGGACCAGGGG-3′ (SEQ IDNO:43) forward PCR primer (34398.f2) 5′-AGCCAGCCTCGCTCTCGG-3′ (SEQ IDNO:44) forward PCR primer (34398.f3) 5′-GTCTGCGATCAGGTCTGG-3′ (SEQ IDNO:45) reverse PCR primer (34398.r1) 5′-GAAAGAGGCAATGGATTCGC-3′ (SEQ IDNO:46) reverse PCR primer (34398.r2) 5′-GACTTACACTTGCCAGCACAGCAC-3′ (SEQID NO:47)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA34398 consensus sequence which had the followingnucleotide sequence

hybridization probe (34398.p1)

5′-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3′ (SEQ ID NO:48)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO344 genes using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO344 [herein designated as DNA40592-1242](SEQ ID NO:41) and the derived protein sequence for PRO344.

The entire nucleotide sequence of DNA40592-1242 is shown in FIG. 17 (SEQID NO:41). Clone DNA40592-1242 contains a single open reading frame withan apparent translational initiation site at nucleotide positions227-229 and ending at the stop codon at nucleotide positions 956-958(FIG. 17). The predicted polypeptide precursor is 243 amino acids long(FIG. 18). Important regions of the native PRO344 amino acid sequenceinclude the signal peptide, the start of the mature protein, and twopotential N-myristoylation sites as shown in FIG. 18. CloneDNA40592-1242 has been deposited with the ATCC and is assigned ATCCdeposit no. ATCC 209492

Analysis of the amino acid sequence of the full-length PRO344polypeptides suggests that portions of them possess significant homologyto various human and murine complement proteins, thereby indicating thatPRO344 may be a novel complement protein.

Example 11

Isolation of cDNA Clones Encoding Human PRO347

A consensus DNA sequence was assembled relative to other EST sequencesas described in Example 1 above. This consensus sequence is hereindesignated DNA39499. Based on the DNA39499 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO347.

PCR primers (forward and reverse) were synthesized as follows:

forward PCR primer 5′-AGGAACTTCTGGATCGGGCTCACC-3′ (SEQ ID NO:51) reversePCR primer 5′-GGGTCTGGGCCAGGTGGAAGAGAG-3′ (SEQ ID NO:52)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA39499 sequence which had the followingnucleotide sequence

hybridization probe

5′-GCCAAGGACTCCTTCCGCTGGGCCACAGGGGAGCACCAGGCCTTC-3′ (SEQ ID NO:53)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO347 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB228).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO347 [herein designated as DNA44176-1244](SEQ ID NO:49) and the derived protein sequence for PRO347.

The entire nucleotide sequence of DNA44176-1244 is shown in FIG. 19 (SEQID NO:49). Clone DNA44176-1244 contains a single open reading frame withan apparent translational initiation site at nucleotide positions123-125 and ending at the stop codon at nucleotide positions 1488-1490(FIG. 19). The predicted polypeptide precursor is 455 amino acids long(FIG. 20). The full-length PRO347 protein shown in FIG. 20 has anestimated molecular weight of about 50,478 daltons and a pI of about8.44. Clone DNA44176-1244 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209532

Analysis of the amino acid sequence of the full-length PRO347polypeptide suggests that portions of it possess significant homology tovarious cysteine-rich secretory proteins, thereby indicating that PRO347may be a novel cysteine-rich secretory protein.

Example 12

Isolation of cDNA Clones Encoding Human PRO354

An expressed sequence tag (EST) DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.) was searched and various ESTsequences were identified which possessed certain degress of homologywith the inter-alpha-trypsin inhibitor heavy chain and with one another.Those homologous EST sequences were then aligned and a consensussequence was obtained. The obtained consensus DNA sequence was thenextended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using homologous EST sequencesderived from both public EST databases (e.g., GenBank) and a proprietaryEST DNA database (LIFESEQ™, Incyte Pharmaceuticals, Palo Alto, Calif.).The extended assembly sequence is herein designated DNA39633. The abovesearches were performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

Based on the DNA39633 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO354. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

PCR primers were synthesized as follows:

forward PCR primer 1 (39633.f1) 5′-GTGGGAACCAAACTCCGGCAGACC-3′ (SEQ IDNO:56) forward PCR primer 2 (39633.f2) 5′-CACATCGAGCGTCTCTGG-3′ (SEQ IDNO:57) reverse PCR primer (39633.r1) 5′-AGCCGCTCCTTCTCCGGTTCATCG-3′ (SEQID NO:58)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA39633 sequence which had the followingnucleotide sequence

hybridization probe

5′-TGGAAGGACCACTTGATATCAGTCACTCCAGACAGCATCAGGGATGGG-3′ (SEQ ID NO:59)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO354 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue (LIB227). The cDNA libraries used to isolate the cDNAclones were constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO354 [herein designated as DNA44192-1246](SEQ ID NO:54) and the derived protein sequence for PRO354.

The entire nucleotide sequence of DNA44192-1246 is shown in FIG. 21 (SEQID NO:54). Clone DNA44192-1246 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 72-74and ending at the stop codon at nucleotide positions 2154-2156 (FIG.21). The predicted polypeptide precursor is 694 amino acids long (FIG.22). The full-length PRO354 protein shown in FIG. 22 has an estimatedmolecular weight of about 77,400 daltons and a pI of about 9.54. CloneDNA44192-1246 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209531.

Analysis of the amino acid sequence of the full-length PRO354polypeptide suggests that it possess significant homology to theinter-alpha-trypsin inhibitor heavy chain protein, thereby indicatingthat PRO354 may be a novel inter-alpha-trypsin inhibitor heavy chainprotein homolog.

Example 13

Isolation of cDNA Clones Encoding Human PRO355

A consensus DNA sequence was assembled relative to other EST sequencesusing BLAST and phrap as described in Example 1 above. This consensussequence is herein designated DNA35702. Based on the DNA35702 consensussequence, oligonucleotides were synthesized: 1) to identify by PCR acDNA library that contained the sequence of interest, and 2) for use asprobes to isolate a clone of the full-length coding sequence for PRO355.

Forward and reverse PCR primers were synthesized as follows:

forward PCR primer 5′-GGCTTCTGCTGTTGCTCTTCTCCG-3′ (SEQ ID NO:62) forwardPCR primer 5′-GTACACTGTGACCAGTCAGC-3′ (SEQ ID NO:63) forward PCR primer5′-ATCATCACAGATTCCCGAGC-3′ (SEQ ID NO:64) reverse PCR primer5′-TTCAATCTCCTCACCTTCCACCGC-3′ (SEQ ID NO:65) reverse PCR primer5′-ATAGCTGTGTCTGCGTCTGCTGCG-3′ (SEQ ID NO:66)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35702 sequence which had the followingnucleotide sequence:

hybridization probe

5′-CGCGGCACTGATCCCCACAGGTGATGGGCAGAATCTGTTTACGAAAGACG-3′ (SEQ ID NO:67)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO355 gene using the probeoligonucleotide. RNA for construction of the cDNA libraries was isolatedfrom human fetal liver tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO355 [herein designated as DNA39518-1247](SEQ ID NO:60) and the derived protein sequence for PRO355.

The entire nucleotide sequence of DNA39518-1247 is shown in FIG. 23 (SEQID NO:60). Clone DNA39518-1247 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 22-24and ending at the stop codon at nucleotide positions 1342-1344 (FIG.23). The predicted polypeptide precursor is 440 amino acids long (FIG.24). The full-length PRO355 protein shown in FIG. 24 has an estimatedmolecular weight of about 48,240 daltons and a pI of about 4.93. Inaddition, regions of interest including the signal peptide, Ig repeatsin the extracellular domain, potential N-glycosylation sites, and thepotential transmembrane domain, are designated in FIG. 24. CloneDNA39518-1247 has been deposited with ATCC and is assigned ATCC depositno. ATCC 209529.

Analysis of the amino acid sequence of the full-length PRO355polypeptide suggests that portions of it possess significant homology tothe CRTAM protein, thereby indicating that PRO355 may be CRTAM protein.

Example 14

Isolation of cDNA Clones Encoding Human PRO357

The sequence expression tag clone no. “2452972” by IncytePharmaceuticals, Palo Alto, Calif. was used to begin a data base search.The extracellular domain (ECD) sequences (including the secretionsignal, if any) of from about 950 known secreted proteins from theSwiss-Prot public protein database were used to search expressedsequence tag (EST) databases which overlapped with a portion of IncyteEST clone no. “2452972”. The EST databases included public EST databases(e.g., GenBank) and a proprietary EST DNA database (LIFESEQ™, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)) as a comparison of the ECD protein sequences to a 6frame translation of the EST sequence. Those comparisons resulting in aBLAST score of 70 (or in some cases 90) or greater that did not encodeknown proteins were clustered and assembled into consensus DNA sequenceswith the program “phrap” (Phil Green, University of Washington, Seattle,Wash.).

A consensus DNA sequence was then assembled relative to other ESTsequences using phrap. This consensus sequence is herein designatedDNA37162. In this case, the consensus DNA sequence was extended usingrepeated cycles of BLAST and phrap to extend the consensus sequence asfar as possible using the sources of EST sequences discussed above.

Based on the DNA37162 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO357. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as ber Ausubel etal., Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

PCR primers were synthesized as follows:

forward primer 1: 5′-CCCTCCACTGCCCCACCGACTG-3′ (SEQ ID NO:70); reverseprimer 1: 5′-CGGTTCTGGGGACGTTAGGGCTCG-3′ (SEQ ID NO:71); and forwardprimer 2: 5′-CTGCCCACCGTCCACCTGCCTCAAT-3′ (SEQ ID NO:72).

Additionally, two synthetic oligonucleotidehybridizationprobes wereconstructed from the consensus DNA37162 sequence which had the followingnucleotide sequences:

hybridization probe 1:

5′ -AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCACC-3′ (SEQ ID NO:73); and

hybridization probe 2:

5′-ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTGGG-3′ (SEQ ID NO:74).

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with aPCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO357 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to Sallhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO357 [herein designated as DNA44804-1248](SEQ ID NO:68) and the derived protein sequence for PRO357.

The entire nucleotide sequence of DNA44804-1248 is shown in FIG. 25 (SEQID NO:68). Clone DNA44804-1248 contains a single open reading frame withan apparent translational initiation site at nucleotide positions137-139 and ending at the stop codon at nucleotide positions 1931-1933(FIG. 25). The predicted polypeptide precursor is 598 amino acids long(FIG. 26). Clone DNA44804-1248 has been deposited with ATCC and isassigned ATCC deposit no. ATCC 209527.

Analysis of the amino acid sequence of the full-length PRO357polypeptide therefore suggests that portions of it possess significanthomology to ALS, thereby indicating that PRO357 may be a novel leucinerich repeat protein related to ALS.

Example 15

Isolation of cDNA Clones Encoding Human PRO715

A proprietary EST DNA database (LIFESEQ™, Incyte Pharmaceuticals, PaloAlto, Calif.) was searched for EST sequences encoding polypeptideshaving homology to human TNF-α. This search resulted in theidentification of Incyte Expressed Sequence Tag No. 2099855.

A consensus DNA sequence was then assembled relative to other ESTsequences using seqext and “phrap” (Phil Green, University ofWashington, Seattle, Wash.). This consensus sequence is hereindesignated DNA52092. Based upon the alignment of the various EST clonesidentified in this assembly, a single EST clone from theMerck/Washington University EST set (EST clone no. 725887, Accession No.AA292358) was obtained and its insert sequenced. The full-lengthDNA52722-1229 sequence was then obtained from sequencing the insert DNAfrom EST clone no. 725887.

The entire nucleotide sequence of DNA52722-1229 is shown in FIG. 27 (SEQID NO:75). Clone DNA52722-1229 contains a single open reading frame withan apparent translational initiation site at nucleotide positions114-116 and ending at the stop codon at nucleotide positions 864-866(FIG. 27). The predicted polypeptide is 250 amino acids long (FIG. 28).The full-length PRO715 protein shown in FIG. 28 has an estimatedmolecular weight of about 27,433 daltons and a pI of about 9.85.

Analysis of the amino acid sequence of the full-length PRO715polypeptide suggests that it possesses significant homology to membersof the tumor necrosis factor family of proteins, thereby indicating thatPRO715 is a novel tumor necrosis factor protein.

Example 16

Isolation of cDNA Clones Encoding Human PRO353

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequences isherein designated DNA36363. The consensus DNA sequence was extendedusing repeated cycles of BLAST and phrap to extend the consensussequence as far as possible using the sources of EST sequences discussedabove. Based on the DNA36363 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO353.

Based on the DNA36363 consensus sequence, forward and reverse PCRprimers were synthesized as follows:

forward PCR primer 5′-TACAGGCCCAGTCAGGACCAGGGG-3′ (SEQ ID NO:79) reversePCR primer 5′-CTGAAGAAGTAGAGGCCGGGCACG-3′ (SEQ ID NO:80).

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA36363 consensus sequence which had the followingnucleotide sequence:

hybridization probe

5′-CCCGGTGCTTGCGCTGCTGTGACCCCGGTACCTCCATGTACCCGG-3′ (SEQ ID NO:81)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO353 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO353 [herein designated as DNA41234-1242](SEQ ID NO:77) and the derived protein sequence for PRO353.

The entire nucleotide sequence of DNA41234-1242 is shown in FIG. 29 (SEQID NO:77). Clone DNA41234-1242 contains a single open reading frame withan apparent translational initiation site at nucleotide positions305-307 and ending at the stop codon at nucleotide positions 1148-1150(FIG. 29). The predicted polypeptide precursor is 281 amino acids long(FIG. 30). Important regions of the amino acid sequence encoded byPRO353 include the signal peptide, corresponding to amino acids 1-26,the start of the mature protein at amino acid position 27, a potentialN-glycosylation site, corresponding to amino acids 93-98 and a regionwhich has homology to a 30 kd adipocyte complement-related proteinprecursor, corresponding to amino acids 99-281. Clone DNA41234-1242 hasbeen deposited with the ATCC and is assigned ATCC deposit no. ATCC209618.

Analysis of the amino acid sequence of the full-length PRO353polypeptides suggests that portions of them possess significant homologyto portions of human and murine complement proteins, thereby indicatingthat PRO353 may be a novel complement protein.

Example 17

Isolation of cDNA Clones Encoding Human PRO361

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA40654. Based on the DNA40654 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO361.

Forward and reverse PCR primers were synthesized as follows:

forward PCR primer 5′-AGGGAGGATTATCCTTGACCTTTGAAGACC-3′ (SEQ ID NO:84)forward PCR primer 5′-GAAGCAAGTGCCCAGCTC-3′ (SEQ ID NO:85) forward PCRprimer 5′-CGGGTCCCTGCTCTTTGG-3′ (SEQ ID NO:86) reverse PCR primer5′-CACCGTAGCTGGGAGCGCACTCAC-3′ (SEQ ID NO:87) reverse PCR primer5′-AGTGTAAGTCAAGCTCCC-3′ (SEQ ID NO:88)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA40654 sequence which had the followingnucleotide sequence

hybridization probe

5′-GCTTCCTGACACTAAGGCTGTCTGCTAGTCAGAATTGCCTCAAAAAGAG-3′ (SEQ ID NO:89)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO361 gene using the probeoligonucleotide. RNA for construction of the cDNA libraries was isolatedfrom human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO361 [herein designated as DNA45410-1250](SEQ ID NO:82) and the derived protein sequence for PRO361.

The entire nucleotide sequence of DNA45410-1250 is shown in FIG. 31 (SEQID NO:82). Clone DNA45410-1250 contains a single open reading frame withan apparent translational initiation site at nucleotide positions226-228 and ending at the stop codon at nucleotide positions 1519-1521(FIG. 31). The predicted polypeptide precursor is 431 amino acids long(FIG. 32). The full-length PRO361 protein shown in FIG. 32 has anestimated molecular weight of about 46,810 daltons and a pI of about6.45. In addition, regions of interest including the transmembranedomain (amino acids 380-409) and sequences typical of the arginasefamily of proteins (amino acids 3-14 and 39-57) are designated in FIG.32. Clone DNA45410-1250 has been deposited with ATCC and is assignedATCC deposit no. ATCC 209621.

Analysis of the amino acid sequence of the full-length PRO361polypeptide suggests that portions of it possess significant homology tothe mucin and/or chitinase proteins, thereby indicating that PRO361 maybe a novel mucin and/or chitinase protein.

Example 18

Isolation of cDNA Clones Encoding Human PRO365

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35613. Based on the DNA35613 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO365.

Forward and reverse PCR primers were synthesized as follows:

forward PCRprimer 5′-AATGTGACCACTGGACTCCC-3′ (SEQ ID NO:92) forward PCRprimer 5′-AGGCTTGGAACTCCCTTC-3′ (SEQ ID NO:93) reverse PCR primer5′-AAGATTCTTGAGCGATTCCAGCTG-3′ (SEQ ID NO:94)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35613 sequence which had the followingnucleotide sequence

hybridization probe

5′-AATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACTG-3′ (SEQ ID NO: 95)

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO365 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO365 [herein designated as DNA46777-1253](SEQ ID NO:90) and the derived protein sequence for PRO365.

The entire nucleotide sequence of DNA46777-1253 is shown in FIG. 33 (SEQID NO:90). Clone DNA46777-1253 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 15-17and ending at the stop codon at nucleotide positions 720-722 (FIG. 33).The predicted polypeptide precursor is 235 amino acids long (FIG. 34).Important regions of the polypeptide sequence encoded by cloneDNA46777-1253 have been identified and include the following: a signalpeptide corresponding to amino acids 1-20, the start of the matureprotein corresponding to amino acid 21, and multiple potentialN-glycosylation sites as shown in FIG. 34. Clone DNA46777-1253 has beendeposited with ATCC and is assigned ATCC deposit no. ATCC 209619.

Analysis of the amino acid sequence of the full-length PRO365polypeptide suggests that portions of it possess significant homology tothe human 2-19 protein, thereby indicating that PRO365 may be a novelhuman 2-19 protein homolog.

Example 19

Use of PRO Polypeptide-Encoding Nucleic Acid as Hybridization Probes

The following method describes use of a nucleotide sequence encoding PROas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO-derived probe to the filters is performed in asolution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate,50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextransulfate at 42° C. for 20 hours. Washing of the filters is performed inan aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 20

Expression of PRO Polypeptides in E. coli

This example illustrates preparation of an unglycosylated form of PRO byrecombinant expression in E. coli.

The DNA sequence encoding PRO is initially amplified using selected PCRprimers. The primers should contain restriction enzyme sites whichcorrespond to the restriction enzyme sites on the selected expressionvector. A variety of expression vectors may be employed. An example of asuitable vector is pBR322 (derived from E. coli; see Bolivar et al.,Gene. 2:95 (1977)) which contains genes for ampicillin and tetracyclineresistance. The vector is digested with restriction enzyme anddephosphorylated. The PCR amplified sequences are then ligated into thevector. The vector will preferably include sequences which encode for anantibiotic resistance gene, a trp promoter, a polyhis leader (includingthe first six STII codons, polyhis sequence, and enterokinase cleavagesite), the PRO coding region, lambda transcriptional terminator, and anargU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO may be expressed in E. coli in a poly-His tagged form, using thefollowing procedure. The DNA encoding PRO is initially amplified usingselected PCR primers. The primers will contain restriction enzyme siteswhich correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) 1on galE rpoHts(htpRts) c1pP(lacIq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA Refolding volumesare chosen so that the fmal protein concentration is between 50 to 100micrograms/ml. The refolding solution is stirred gently at 4° C. for12-36 hours. The refolding reaction is quenched by the addition of TFAto a final concentration of 0.4% (pH of approximately 3). Before furtherpurification of the protein, the solution is filtered through a 0.22micron filter and acetonitrile is added to 2-10% final concentration.The refolded protein is chromatographed on a Poros R1/H reversed phasecolumn using a mobile buffer of 0.1% TFA with elution with a gradient ofacetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbanceare analyzed on SDS polyacrylamide gels and fractions containinghomogeneous refolded protein are pooled. Generally, the properlyrefolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO polypeptide are pooled andthe acetonitrile removed using a gentle stream of nitrogen directed atthe solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14M sodium chloride and 4% mannitol by dialysis or by gel filtration usingG25 Superfine (Pharmacia) resins equilibrated in the formulation bufferand sterile filtered.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 21

Expression of PRO Polypeptides in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof PRO by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO DNA is ligated into pRK5with selected restriction enzymes to allow insertion of the PRO DNAusing ligation methods such as described in Sambrook et al., supra. Theresulting vector is called pRK5-PRO.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PRODNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μlof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

In another embodiment, PRO can be expressed in CHO cells. The pRK5-PROcan be transfected into CHO cells using known reagents such as CaPO₄ orDEAE-dextran. As described above, the cell cultures can be incubated,and the medium replaced with culture medium (alone) or medium containinga radiolabel such as ³⁵S-methionine. After determining the presence ofPRO polypeptide, the culture medium may be replaced with serum freemedium. Preferably, the cultures are incubated for about 6 days, andthen the conditioned medium is harvested. The medium containing theexpressed PRO can then be concentrated and purified by any selectedmethod.

Epitope-tagged PRO may also be expressed in host CHO cells. The PRO maybe subcloned out of the pRK5 vector. The subclone insert can undergo PCRto fuse in frame with a selected epitope tag such as a poly-his tag intoa Baculovirus expression vector. The poly-his tagged PRO insert can thenbe subcloned into a SV40 driven vector containing a selection markersuch as DHFR for selection of stable clones. Finally, the CHO cells canbe transfected (as described above) with the SV40 driven vector.Labeling may be performed, as described above, to verify expression. Theculture medium containing the expressed poly-His tagged PRO can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 22

Expression of PRO in Yeast

The following method describes recombinant expression of PRO in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO can subsequently be isolated and purified by removingthe yeast cells from the fermentation medium by centrifugation and thenconcentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 23

Expression of PRO in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO can then be purified, for example, byNi²⁺-chelate affinity chromatography as follows. Extracts are preparedfrom recombinant virus-infected Sf9 cells as described by Rupert et al.,Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspendedin sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA;10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 secondson ice. The sonicates are cleared by centrifugation, and the supernatantis diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO are pooled and dialyzedagainst loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO can beperformed using known chromatography techniques, including for instance,Protein A or protein G column chromatography.

Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 24

Preparation of Antibodies that Bind PRO

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO, fusion proteins containing PRO, andcells expressing recombinant PRO on the cell surface. Selection of theimmunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the PRO immunogen emulsified incomplete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO. Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 25

Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 26

Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 27

Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem. 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 28

Gene Amplification

This example shows that the PRO327-, PRO344-, PRO347-, PRO357- andPRO715-encoding genes are amplified in the genome of certain human lung,colon and/or breast cancers and/or cell lines. Amplification isassociated with overexpression of the gene product, indicating that thepolypeptides are useful targets for therapeutic intervention in certaincancers such as colon, lung, breast and other cancers. Therapeuticagents may take the form of antagonists of PRO327, PRO344, PRO347,PRO357 aor PRO715 polypeptide, for example, murine-human chimeric,humanized or human antibodies against a PRO327, PRO344, PRO347, PRO357or PRO715 polypeptide. These amplifications also are useful asdiagnostic markers for the presence of a specific type of tumor type.

The starting material for the screen was genomic DNA isolated from avariety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TaqMan™) and real-time quantitative PCR(for example, ABI Prizm 7700 Sequence Detection System™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding PRO327, PRO344, PRO347, PRO357 orPRO715 is over-represented in any of the primary lung or colon cancersor cancer cell lines or breast cancer cell lines that were screened. Theprimary lung cancers were obtained from individuals with tumors of thetype and stage as indicated in Table 9. An explanation of theabbreviations used for the designation of the primary tumors listed inTable 9 and the primary tumors and cell lines referred to throughoutthis example are given below.

The results of the TaqMan™ are reported in delta (Δ) Ct units. One unitcorresponds to 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTaqMan™ fluorescent probe derived from the PRO327-, PRO344-, PRO347-,PRO357- or PRO715-encoding gene. Regions of PRO327, PRO344, PRO347,PRO357 or PRO715 which are most likely to contain unique nucleic acidsequences and which are least likely to have spliced out introns arepreferred for the primer and probe derivation, e.g., 3′-untranslatedregions. The sequences for the primers and probes (forward, reverse andprobe) used for the PRO327, PRO344, PRO347, PRO357 or PRO715 geneamplification analysis were as follows:

PRO327 (DNA381 13-1230) forward 5′-CTCAAGAAGCACGCGTACTGC-3′ (SEQ IDNO:96) probe 5′-CCAACCTCAGCTTCCGCCTCTACGA-3′ (SEQ ID NO:97) reverse5′-CATCCAGGCTCGCCACTG-3′ (SEQ ID NO :98) PRO344 (DNA40592-1242) forward5′-TGGCAAGGAATGGGAACAGT-3′ (SEQ ID NO:99) probe5′-ATGCTGCCAGACCTGATCGCAGACA-3′ (SEQ ID NO:100) reverse5′-GGGCAGAAATCCAGCCACT-3′ (SEQ ID NO:101) PRO347 (DNA44176-1244) forward5′-CCCTTCGCCTGCTTTTGA-3′ (SEQ ID NO:102) probe5′-GCCATCTAATTGAAGCCCATCTTCCCA-3′ (SEQ ID NO:103) reverse5′-CTGGCGGTGTCCTCTCCTT-3′ (SEQ ID NO:104) PRO357 (DNA44804-1248) forward5′-CCTCGGTCTCCTCATCTGTGA-3′ (SEQ ID NO:105) probe5′-TGGCCCAGCTGACGAGCCCT-3′ (SEQ ID NO:106) reverse5′-CTCATAGGCACTCGGTTCTGG-3′ (SEQ ID NO:107) PRO715  (DNA52722-1229)forward 5′-TGGCTCCCAGCTTGGAAGA-3′ (SEQ ID NO:108) probe5′-CAGCTCTTGGCTGTCTCCAGTATGTACCCA-3′ (SEQ ID NO:109) reverse5′-GATGCCTCTGTTCCTGCACAT-3′ (SEQ ID NO:110)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers are used to generate an amplicon typical of a PCR reaction. Athird oligonucleotide, or probe, is designed to detect nucleotidesequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700TM Sequence Detection. The system consists ofa thermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

5′ Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer DNA results to normal human DNA results.

Table 9 describes the stage, T stage and N stage of various primarytumors which were used to screen the PRO327, PRO344, PRO347, PRO357 andPRO715 compounds of the invention.

TABLE 9 Primary Lung and Colon Tumor Profiles Primary Tumor Stage StageOther Stage Dukes Stage T Stage N Stage Human lung tumor AdenoCa(SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0 Human lung tumorAdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728)[LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Humanlung tumor Aden/SqCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa(SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumorAdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa(SRCC735)[LT13] IB T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2N0 Human lung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumorSqCCa (SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18]IB T2 N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lungtumor LCCa (SRCC741) [LT2] IIB T3 N1 Human lung AdenoCa (SRCC811) [LT22]1A T1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Human colonAdenoCa (SRCC743) [CT3] B pT3 N0 Human colon AdenoCa (SRCC744) [CT8] BT3 N0 Human colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human colon AdenoCa(SRCC746) [CT12] MO, R1 B T3 N0 Human colon AdenoCa (SRCC747) [CT14]pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa(SRCC75O) [CT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 BpT3 N0 Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa(SRCC753) [CT5] G2 Cl pT3 pN0 Human colon AdenoCa (SRCC754) [CT6] pMO,RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0 Humancolon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa (SRCC757)[CT11] B T3 N0 Human colon AdenoCa (SRCC758) [CT18] MO, RO B pT3 pN0

DNA Preparation

DNA was prepared from cultured cell lines, primary tumors, normal humanblood. The isolation was performed using purification kit, buffer setand protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell culture lysis

Cells were washed and trypsinized at a concentration of 7.5×10⁸ per tipand pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂O to a final concentration of 20 mg/ml and equilibratedat 4° C. 10 ml of G2 Buffer was prepared by diluting Qiagen RNAse Astock (100 mg/ml) to a final concentration of 200 μg/ml.

Buffer C1 (10 ml, 4° C.) and ddH20 (40 ml, 4° C.) were then added to the10 ml of cell suspension, mixed by inverting and incubated on ice for 10minutes. The cell nuclei were pelleted by centrifuging in a Beckmanswinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30-60 minutes, pelleting at 3000×g for 10 min., 4°C.).

Solid human tumor sample preparation and lysis

Tumor samples were weighed and placed into 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂O, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

Human blood preparation and lysis

Blood was drawn from healthy volunteers using standard infectious agentprotocols and citrated into 10 ml samples per tip. Quiagen protease wasfreshly prepared by dilution into 6.25 ml cold ddH2O to a finalconcentration of 20 mg/ml and stored at 4° C. G2 buffer was prepared bydiluting RNAse A to a final concentration of 200 μg/ml from 100 mg/mlstock. The blood (10 ml) was placed into a 50 ml conical tube and 10 mlC1 buffer and 30 ml ddH₂O (both previously equilibrated to 4° C.) wereadded, and the components mixed by inverting and held on ice for 10minutes. The nuclei were pelleted with a Beckman swinging bucket rotorat 2500 rpm, 4° C. for 15 minutes and the supernatant discarded. With avortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and 6 mlddH₂O (4° C). Vortexing was repeated until the pellet was white. Thenuclei were then suspended into the residual buffer using a 200 μl tip.G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30-60 minutes, pelleting at 3000×g for 10min., 4° C.).

Purification of cleared lysates

(1) Isolation of genomic DNA:

Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10ml QBT buffer. QF elution buffer was equilibrated at 50° C. The sampleswere vortexed for 30 seconds, then loaded onto equilibrated tips anddrained by gravity. The tips were washed with 2×15 ml QC buffer. The DNAwas eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 mlQF buffer (50° C.). Isopropanol (10.5 ml) was added to each sample, thetubes covered with parafin and mixed by repeated inversion until the DNAprecipitated. Samples were pelleted by centrifugation in the SS-34 rotorat 15,000 rpm for 10 minutes at 4° C. The pellet location was marked,the supernatant discarded, and 10 ml 70% ethanol (4° C.) was added.Samples were pelleted again by centrifugation on the SS-34 rotor at10,000 rpm for 10 minutes at 4° C. The pellet location was marked andthe supernatant discarded. The tubes were then placed on their side in adrying rack and dried 10 minutes at 37° C., taking care not to overdrythe samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) andplaced at 50° C. for 1-2 hours. Samples were held overnight at 4° C. asdissolution continued. The DNA solution was then transferred to 1.5 mltubes with a 26 gauge needle on a tuberculin syringe. The transfer wasrepeated 5× in order to shear the DNA. Samples were then placed at 50°C. for 1-2 hours.

(2) Quantitation of penomic DNA and preparation for gene amplificationassay:

The DNA levels in each tube were quantified by standard A₂₆₀, A₂₈₀spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) using the0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A₂₆₀/A₂₈₀ratios were in the range of 1.8-1.9. Each DNA samples was then dilutedfurther to approximately 200 ng/ml in TE (pH 8.5). If the originalmaterial was highly concentrated (about 700 ng/μl), the material wasplaced at 50° C. for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material(20-600 ng/ml) using the manufacturer's guidelines as modified below.This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes. The Hoechst dye working solution(#H33258, 10 μl, prepared within 12 hours of use) was diluted into 100ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

The fluorometricly determined concentration was then used to dilute eachsample to 10 ng/μl in ddH₂O. This was done simultaneously on alltemplate samples for a single TaqMan plate assay, and with enoughmaterial to run 500-1000 assays. The samples were tested in triplicatewith Taqman™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8-9 plates or 64 tests.

Gene amplification assay:

The PRO327, PRO344, PRO347, PRO357 and PRO715 compounds of the inventionwere screened in the following primary tumors and the resulting ΔCtvalues greater than or equal to 1.0 are reported in Table 10.

TABLE 10 ΔCt values in primary tumor and cell lines models PrimaryTumors or Cell Lines PRO327 PRO344 PRO347 PRO357 PRO715 LT1 — — 1.035 —1.625 LT1a 1.045 — 1.865 1.18 1.045 1.0 2.47 1.93 LT3 1.135 — 1.325 2.93— 1.2 LT6 1.395 — 1.945 2.6 — 1.42 3.18 LT9 — — 2.645 3.47 1.005 2.91LT10 1.305 — 1.845 3.42 1.125 1.13 3.51 LT11 1.53 1.52 1.395 1.185 1.751.35 2.875 1.12 LT12 2.99 1.2 1.425 1.225 1.63 2.15 1.73 2.225 1.11 1.14LT13 2.48 1.81 2.035 1.585 2.29 1.69 1.175 2.28 1.665 1.83 1.05 1.151.31 LT15 3.99 1.62 1.615 2.205 2.33 2.89 1.33 2.73 2.445 1.89 1.27 1.891.44 LT16 1.16 1.13 — 2.605 1.2 2.65 1.09 1.1 LT17 1.76 1.46 1.24 1.2751.95 1.09 2.855 1.33 1.01 LT18 — — — 2.455 1.14 LT19 3.58 2.47 1.8352.295 2.38 1.35 2.645 LT21 — 1.09 1.14 2.675 — CT2 3.645 1.84 2.1 2.011.675 1.605 1.605 CT3 1.125 — 1.01 — 1.135 1.105 CT8 1.645 — 1.3 1.11.285 1.345 CT10 2.535 — — 1.42 2.155 1.785 CT12 1.885 — — — — CT142.515 1.16 1.39 1.5 1.265 1.45 1.575 CT15 1.305 1.17 1.3 1.25 1.5851.475 CT16 1.475 — 1.33 1.05 1.095 1.055 1.475 CT17 1.715 — — — 1.2451.375 CT1 1.375 1.245 1.045 1.045 1.285 1.6 1.085 CT4 2.225 1.465 —1.275 1.375 2.23 1.165 CT5 2.505 1.515 1.625 1.695 1.975 1.985 2.071.715 CT6 2.285 — — 1.085 1.305 1.73 1.245 CT7 — — — 1.735 1.005 1.651.025 CT9 1.585 — — — 1.0 CT11 3.335 1.355 1.315 1.835 2.185 1.525 2.54CT18 1.075 — — — 1.69 SRCC771 1.65 — — — — (H157) SRCC772 2.23 — — — —(H441) SRCC773 1.12 — — — — (H460) SRCC774 1.18 — — — — (SKMES-1)SRCC777 2.24 — — — — (SW620) SRCC778 1.01 — — — — (Colo320) SRCC830 1.23— — — — (HCC2998) SRCC831 1.61 — — — — (KM12) SRCC832 1.02 — — — —(H522) SRCC833 1.11 — — — — (H810)

PRO327

PRO327 (DNA38113-1230) was reexamined along with selected tumors fromthe above initial screen with framework mapping. Table 11 describes theframework markers that were employed in association with PRO327(DNA38113-1230). The framework markers are located approximately every20 megabases along Chromosome 19, and are used to control aneuploidy.The ΔCt values for the described framework markers along Chromosome 19relative to PRO327 (DNA38113-1230) are indicated for selected tumors inTable 13.

PRO327 (DNA38113-1230) was also reexamined along with selected tumorsfrom the above initial screen with epicenter mapping. Table 12 describesthe epicenter markers that were employed in association with PRO327(DNA38113-1230). These markers are located in close proximity toDNA38113-1230 and are used to assess the amplification status of theregion of Chromosome 19 in which DNA38113-1230 is located. The distancebetween markers is measured in centirays (cR), which is a radiationbreakage unit approximately equal to a 1% chance of a breakage betweentwo markers. One cR is very roughly equivalent to 20 kilobases.

Table 14 indicates the ΔCt values for results of epicenter mappingrelative to DNA38113-1230, indicating the relative amplification in theregion more immediate to the actual location of DNA38113-1230 alongChromosome 19.

TABLE 11 Framework Markers Along Chromosome 19 Map Position onChromosome 19 Stanford Human Genome Center Marker Name S12 AFMa107xc9S50 SHGC-31335 S105 SHGC-34102 S155 SHGC-16175

TABLE 12 Epicenter Markers Along Chromosome 19 used for DNA38113-1230Map Position Stanford Human Genome Distance to next Marker on Chromosome19 Center Marker Name (cR¹) S42 WI-7289  5 S43 SHGC-32638 28 S44 SHGC-11753²  7 DNA38113-1230 — — S45 SHGC-14810 37 S46 AFM214YF6 15 S48SHGC-36583 —

TABLE 13 Amplification of framework markers relative to DNA38113-1230(ΔCt) Framework Markers DNA38113- Tumor S12 1230 S50 S105 S155 LT1 0.16−0.15 0.06 −0.42 0.11 LT1a 0.05 0.57 −0.27 0.17 0.40 LT2 0.48 0.57 0.410.52 0.13 LT3 0.27 0.77 0.83 0.11 0.50 LT4 0.48 0.08 0.67 0.20 0.56 LT60.72 0.33 0.74 0.32 0.35 LT7 0.82 0.29 0.85 0.95 0.95 LT9 0.72 −0.190.61 0.19 0.64 LT10 0.82 1.45 0.98 0.62 0.53 CT2 0.25 2.94 0.29 0.37−0.02 CT3 −0.17 1.23 −0.10 0.34 −0.28 CT8 0.13 1.45 0.57 0.18 −0.16 CT100.15 1.72 0.51 −0.01 −0.81 CT12 0.13 1.60 0.57 0.41 0.20 CT14 0.40 2.030.39 0.45 0.36 CT15 −0.23 0.68 −0.30 −0.06 0.56 CT16 0.38 1.07 0.31 0.240.04 CT17 0.25 0.50 0.71 0.32 0.09

TABLE 14 Amplification of epicenter markers relative to DNA38113-1230(ΔCt) Tu- DNA38113- mor S41 S42 S43 S44 1230 S45 S46 S48 LT1 −1.03 −0.25−0.18 −0.11 −0.31 0.13 0.26 0.29 LT1a 0.14 −0.30 −0.11 −0.01 0.21 −0.440.45 −0.30 LT2 0.03 0.06 0.06 0.12 0.14 0.16 0.11 0.65 LT3 −1.08 −0.08−0.01 0.11 0.43 −0.37 0.33 0.56 LT4 0.66 −0.14 −0.48 −0.79 −0.28 −0.310.04 0.09 LT6 −0.88 −0.08 −0.12 −1.00 0.20 −0.43 0.48 0.63 LT7 0.65−0.19 −0.19 −0.04 0.04 −0.42 0.43 0.57 LT9 0.66 −0.26 −0.01 −0.14 −0.06−0.31 −16.48 0.16 LT10 1.16 −0.30 −0.11 −0.31 0.13 −0.33 0.34 0.50 LT110.46 0.01 −0.04 −0.86 0.67 0.23 0.24 −0.57 LT12 1.39 −0.01 −0.22 −1.331.57 −0.25 0.26 0.07 LT13 1.62 −0.03 0.00 −0.08 1.22 −0.08 0.48 0.14LT15 1.09 0.20 0.47 0.62 2.47 0.38 0.01 0.44 LT16 1.51 0.04 −0.04 0.292.23 0.51 0.50 0.90 LT17 2.12 0.23 0.11 0.20 1.02 0.45 0.46 −0.41 LT181.80 −0.11 0.07 −0.70 0.9 0.10 0.00 −0.02 LT22 −0.12 0.06 0.41 −0.11−0.06 0.34 0.03 0.52 CT1 −0.09 0.33 0.11 0.22 1.38 0.09 −0.25 −0.10 CT21.76 0.04 0.30 0.65 2.94 0.18 −0.04 0.01 CT3 1.10 −0.31 −0.24 0.16 1.23−0.64 0.78 −0.17 CT4 1.63 0.22 0.32 −0.72 2.23 −0.04 0.44 0.72 CT5 2.220.02 0.21 0.10 2.51 0.02 0.18 0.24 CT6 0.48 0.20 0.22 −0.63 2.29 0.030.14 0.97 CT7 0.93 0.20 0.32 0.14 0.95 −0.01 0.20 0.54 CT8 1.15 −0.500.14 0.15 1.45 −0.31 0.54 0.07 CT9 0.82 0.38 0.64 −0.71 1.59 1.04 0.260.93 CT10 1.57 −0.41 −0.03 −0.14 1.72 −0.27 0.04 0.10 CT11 1.49 −0.050.07 0.01 3.34 0.54 0.28 0.88 CT12 0.89 −0.09 −0.01 −0.62 1.6 −0.07 1.160.92 CT14 2.16 0.32 0.37 0.47 2.03 −0.07 1.21 0.44 CT15 0.64 −0.52 −0.21−0.12 0.68 −0.61 1.01 0.32 CT16 1.75 −0.31 0.28 0.47 1.07 0.04 1.01−0.29 CT17 0.77 −0.18 0.13 −0.04 0.5 −0.27 0.93 0.31 CT18 0.91 0.05 0.140.60 1.08 0.22 −0.59 0.61

PRO715 (DNA52722-1229)

PRO715 was also reexamined with both framework and epicenter mapping.Table 15 indicates the chromosomal localizations of the frameworkmarkers that were used for the procedure. The framework markers arelocated approximately every 20 megabases and were used to controlaneuploidy. Table 16 indicates the epicenter mapping markers that wereused in the procedure. The epicenter markers were located in closeproximity to DNA52722-1229 and are used to determine the relative DNAamplification in the immediate vicinity of DNA52722-1229. The distancebetween individual markers is measured in centirays, which is aradiation breakage unit approximately equal to a 1% chance of a breakagebetween two markers. One cR is very roughly equivalent to about 20kilobases. In Table 16, “BAC” means bacterial artificial chromosome. Theends of a BAC clone which contained the gene of interest were sequenced.TaqMan primers and probes were made from this sequence, which areindicated in the table. BAC clones are typically 100 to 150 Kb, so theseprimers and probes can be used as nearby markers to probe DNA fromtumors. In Table 16, the marker SHGC-31370 is the marker found to be theclosest to the location on chromosome 17 where DNA52722-1229 maps.

TABLE 15 Framework Markers Used Along Chromosome 17 for DNA52722-1229Stanford Human Genome Center Map Position on Chromosome 17 Marker NameQ4 SHGC-31242 Q52 SHGC-35988 Q110 AFM200zf4 Q169 SHGC-32689 Q206SHGC-11717 Q232 SHGC-32338

TABLE 16 Epicenter Markers Used on Chromosome 17 in Vicinity ofDNA52722-1229 Map Position on Stanford Human Genome Marker Distance tonext Chromosome 17 Name Marker (cR) Q33 SHGC-35547 18 cR to Q34120F17FOR1 Marker from forward end of BAC sequence 120F17FOR2 Markerfrom forward end of BAC sequence DNA52722-1229 — 120F17REV1 Marker fromreverse end of BAC sequence 120F17REV2 Marker from reverse end of BACsequence Q34 SHGC-31370

Table 17 indicates the ΔCt values of the above described frameworkmarkers along chromosome 17 relative to DNA52722-1229 for selectedtumors.

TABLE 17 Amplification of Framework Markers Relative to DNA52722-1229Framework Marker DNA5272 Tumor Q4 Q52 2-1229 Q110 Q169 Q206 Q232 LT10.02 −0.50 −0.04 0.05 −0.32 −0.21 −0.34 LT1a −0.01 −0.34 0.64 0.23 −0.20−0.25 −0.15 LT2 0.25 0.15 0.19 0.05 −0.16 −0.14 −0.09 LT3 −0.08 −0.200.54 0.56 −0.06 0.32 0.05 LT4 −0.32 −0.45 0.31 0.19 −0.06 −0.12 0.04 LT6−0.21 −0.38 0.31 0.13 −0.08 −0.30 0.01 LT7 −0.66 −1.02 0.02 0.62 −0.200.06 0.16 LT9 −0.03 −0.29 0.46 1.20 −1.75 −0.22 −0.13 LT10 −0.16 −0.090.58 0.11 0.01 −0.33 −0.45 LT11 −0.14 0.29 1.03 0.04 0.30 0.52 0.17 LT12−0.25 −0.68 0.72 0.65 0.86 0.97 0.58 LT13 0.20 0.00 1.37 −0.15 −0.040.25 −0.01 LT15 0.11 −0.39 1.75 0.00 −0.02 0.43 −0.19 LT16 −0.07 −0.561.11 0.22 0.19 0.68 −0.55 LT17 0.41 −0.09 1.14 0.27 0.22 0.73 0.07 LT180.14 −0.22 1.04 0.27 0.35 0.48 −0.03 LT22 −0.07 −0.73 0.00 0.13 −0.020.41 0.05 CT2 0.12 −0.47 1.29 −0.19 0.32 — 0.18 CT3 0.05 0.17 1.06 −0.410.05 — −0.06 CT8 0.44 0.14 1.08 0.02 −0.04 — −0.11 CT10 0.35 0.26 1.60−0.05 0.00 — −0.02 CT12 −0.15 −0.46 0.52 −0.13 0.02 — −0.20 CT14 0.26−0.59 1.05 −0.01 0.68 — 0.48 CT15 0.55 −0.51 1.36 −0.69 0.11 — −0.16CT16 0.09 −0.14 1.06 0.00 0.00 — −0.15 CT17 0.40 −0.16 1.00 −0.47 0.04 —−0.29

Table 18 indicates the ΔCt values for the indicated epicenter markers,indicating the relative amplification along chromosome 17 in theimmediate vicinity of DNA52722-1229.

TABLE 18 Amplification of Epicenter Markers Relative to DNA52722-1229Epicenter marker 120F17FOR 120F17FOR DNA5272 Tumor Q33 1 2 2-1229120F17REV1 120F17REV2 Q34 LT1 −0.18 0.11 0.00 0.20 −0.08 0.07 −0.36 LT1a0.32 −0.06 0.00 0.68 −0.09 −0.20 0.32 LT2 0.06 0.14 0.00 0.27 −0.29 0.16−0.16 LT3 0.08 −2.06 0.00 0.16 −0.84 −0.38 −0.16 LT4 — — — — — — — LT6 —— — — — — — LT7 −0.20 −0.51 0.00 0.23 −0.63 −0.37 −0.41 LT9 0.08 −0.170.00 0.59 0.02 −0.66 −0.01 LT10 0.09 0.05 0.00 0.59 −0.22 −0.12 0.36LT11 0.75 0.09 0.00 1.07 0.43 −0.01 0.63 LT12 0.00 −0.45 0.00 0.63 −0.49−0.82 0.18 LT13 0.72 −0.02 0.00 1.29 0.04 0.02 0.66 LT15 0.75 0.11 0.001.33 0.15 −0.19 0.90 LT16 0.34 −0.41 0.00 1.11 −0.39 −0.89 0.15 LT171.06 0.29 0.00 1.13 −0.26 −0.12 0.90 LT18 0.66 0.11 0.00 1.21 −0.28 0.110.47 LT19 −0.09 −0.37 0.00 0.12 −0.53 −0.48 −0.53 CT1 0.50 0.14 0.001.22 0.27 0.43 0.72 CT2 0.69 −0.47 0.00 0.95 −0.72 −0.17 0.77 CT3 0.870.08 0 1.19 −0.06 0.74 0.97 CT4 0.45 −0.11 0 1.26 0.43 0.38 0.79 CT50.36 −0.39 0 1.79 −0.48 0.09 0.95 CT6 0.41 0.08 0 1.71 −0.21 0.57 0.47CT7 0.40 0.18 0 1.19 0.31 0.40 0.54 CT8 0.48 0.17 0 0.93 0.23 0.47 0.72CT10 0.72 0.15 0 1.86 0.81 0.67 0.97 CT11 0.80 −0.09 0 2.29 0.20 0.250.85 CT12 0.01 −0.55 0 0.49 −0.43 −0.09 0.11 CT14 0.22 −0.36 0 1.05 0.630.41 0.40 CT15 1.06 −0.04 0 1.27 0.74 0.98 1.13 CT16 0.84 0.06 0 1.030.26 0.40 0.91 CT17 0.80 0.04 0 0.95 0.78 1.29 0.90 CT18 0.34 0.13 01.06 0.06 0.34 0.50

PRO357 (DNA44804-1248)

PRO357 was reexamined with selected tumors from the above initial screenwith framework mapping. Table 19 indicate the chromosomal mapping of theframework markers that were used in the present example. The frameworkmarkers are located approximately every 20 megabases and were used tocontrol aneuploidy.

PRO357 was also examined with epicenter mapping. The markers indicatedin Table 20 are located in close proximity (in the genome) toDNA44804-1248 and are used to assess the relative amplification in theimmediate vicinity of chromosome 16 wherein DNA44804-1248 is located.The distance between individual markers is measured in centirays (cR),which is a radiation breakage unit approximately equal to a 1% chance ofa breakage between the two markers. One cR is very roughly equivalent to20 kilobases. The marker SHGC-6154 is the marker found to be the closestto the location on chromosome 16 where DNA44804-1248 maps.

TABLE 19 Framework markers for DNA44804-1248 Stanford Human GenomeCenter Map position on chromosome 16 Marker Name P7 SHGC-2835 P55SHGC-9643 P99 GATA7B02 P154 SHGC-33727 P208 SHGC-13577

TABLE 20 Epicenter markers for DNA44804-1248 along chromosome 16 Mapposition on Stanford Human Genome Distance to next chromosome 16 CenterMarker Name Marker (cR) P1 AFMA139WG1  6 P3 SHGC-32420       170 (gap)P4 SHGC-14817 40 P5 SHGC-12265  4 P6 SHGC-6154 33 DNA44804-1248 — — P7SHGC-2835 10 P8 SHGC-2850  9 P9 AFM297yg5 67 P15 CHLC.GATA70B04 —

The ΔCt values of the above described framework markers along chromosome16 relative to DNA44804-1248 is described in Table 21.

TABLE 21 Amplification of Framework Markers relative to DNA44804-1248(ΔCt) Framework marker DNA44804- Tumor 1248 P7 P55 P99 P154 208 LT1 0.250.22 −0.17 0.42 0.04 0.43 LT1a 0.90 0.09 −0.10 −0.38 0.29 0.93 LT2 −0.160.03 0.19 −0.18 0.18 0.54 LT3 1.15 0.68 0.57 −0.34 −0.03 0.86 LT4 0.190.58 0.36 −0.31 0.08 1.14 LT6 0.28 0.27 −0.11 −0.74 −0.13 0.22 LT7 0.580.63 0.14 0.82 0.09 −0.21 LT9 0.68 0.63 0.14 0.82 0.09 −0.21 LT10 1.210.52 0.40 −0.39 −0.15 0.77 LT11 1.71 −0.79 1.31 0.73 −0.08 0.90 LT121.96 −0.95 0.94 0.00 −0.63 0.18 LT13 2.32 −0.97 0.94 0.88 −0.04 0.70LT15 3.01 −0.54 0.60 0.12 0.14 1.15 LT16 0.67 −0.27 0.57 −0.39 0.08 1.04LT17 1.64 0.25 1.10 0.28 0.10 0.23 LT18 0.34 0.09 0.51 0.33 −0.20 −0.09LT19 3.03 −0.82 0.63 0.06 0.09 0.55 LT21 1.33 −1.19 1.01 0.11 0.34 0.07

Table 22 indicates the ΔCt values for the results of epicenter mappingrelative to DNA44804-1248, indicating the relative amplification in theregion more immediate to the actual location of DNA44804-1248 alongchromosome 16.

TABLE 22 Amplification of epicenter markers relative to DNA44804-1248Epicenter marker DNA4480 Tumor P1 P3 P4 P5 P6 4-1248 P7 P8 P9 P15 LT10.31 −0.30 0.65 0.05 −0.33 0.16 −0.41 0.20 0.1 0.17 LT1a −0.23 −17.670.97 −0.65 −1.83 0.56 −0.65 −0.28 −0.27 −0.07 LT2 0.18 −0.06 0.33 −0.11−0.38 −0.32 −1.08 −0.31 −0.53 −0.05 LT3 0.00 0.25 1.07 −0.23 −0.11 0.70−0.71 −0.12 −0.17 −0.01 LT4 0.07 −0.25 0.55 −1.15 −1.78 −0.09 −0.82−0.07 −0.34 −0.07 LT6 0.24 0.07 0.48 −0.55 −0.34 −0.07 −1.33 −0.41 −0.7−0.27 LT7 0.07 −0.07 0.61 −0.19 −0.36 0.29 −0.96 −0.09 −0.26 −0.08 LT90.16 −0.16 0.64 −0.33 −0.14 0.43 −1.01 −0.19 −0.36 −0.21 LT10 0.47 0.76−0.30 0.80 −0.09 0.00 −0.85 −0.17 −0.28 −0.07 LT11 0.14 0.14 0.96 −0.020.37 1.27 −0.23 0.09 −0.33 −0.07 LT12 −0.12 −0.04 0.84 −1.52 −0.28 1.42−0.39 −0.38 −1.21 −0.25 LT13 0.41 −0.02 1.19 −0.34 0.14 1.67 −0.87 −0.22−0.72 −0.33 LT15 0.01 0.21 1.30 −0.48 −0.35 2.36 −0.96 −0.36 −0.54 −0.22LT16 −0.38 −0.07 0.41 −0.32 −1.22 −0.08 −0.45 −0.25 −0.52 −0.31 LT170.36 0.23 1.39 −1.39 01.37 1.17 −0.39 −0.13 0.52 0.01 LT18 0.17 −0.270.04 −0.04 0.18 −0.39 −0.59 −0.25 −0.21 −0.22 LT19 0.11 −0.02 1.27 −0.121.27 2.49 −0.30 −0.36 −0.82 −0.40 LT21 0.28 −0.18 0.85 0.09 0.66 0.85−0.49 −0.35 −0.27 −0.16

CONCLUSION

The ΔCt values for the above DNAs in a variety of tumors are reported. AΔCt of >1 was typically used as the threshold value for amplificationscoring, as this represents a doubling of gene copy. The above dataindicates that significant amplification of the tested nucleic acidsoccurred in primary lung tumors and/or primary colon tumors:Amplification has been confirmed by framework mapping. The frameworkmarkers analysis reports the relative amplification of particularchromosomal regions in the indicated tumors, while the epicenter markersanalysis gives a more precise reading of the relative amplification inthe region immediately in the vicinity of the gene of interest.

Amplification has been confirmed by epicenter mapping and the dataevidenced significant amplification in primary colon tumors and/orprimary lung tumors: Amplification of the closest known epicentermarkers does not occur to a greater extent than that of the DNAs tested.This strongly suggests that the DNAs tested are responsible for theamplification of the particular region on the respective chromosome.

Because amplification of the DNAs tested occurs in various lung andcolon tumors, it is highly probable that these DNAs play a significantrole in tumor formation or growth. As a result, antagonists (e.g.,antibodies) directed against the proteins encoded by the DNAs testedwould be expected to have utility in cancer therapy and as usefuldiagnostic reagents. The polypeptides encoded by the DNAs tested haveutility as diagnostic markers for determining the presence of tumorcells in lung and/or colon tissue samples. The nucleic acid sequencesencoding these polypeptides have utility as sources of nucleic acidprobes for carrying out the above diagnostic procedures.

Example 29

Ability of PRO241 to Stimulate the Release of Proteoglycans fromCartilage (Assay 97)

The ability of PRO241 to stimulate the release of proteoglycans fromcartilage tissue was tested as follows. A positive result in this assayevidences that the polypeptide is expected to be useful in thetherapeutic treatment of various cartilage and/or bone injuries ordisorders including, for example, arthritis.

The metacarphophalangeal joint of 4-6 month old pigs was asepticallydissected, and articular cartilage was removed by free hand slicingbeing careful to avoid the underlying bone. The cartilage was minced andcultured in bulk for 24 hours in a humidified atmosphere of 95% air, 5%CO₂ in serum free (SF) media (DME/F12 1:1) woth 0.1% BSA and 100 U/mlpenicillin and 100 μg/ml streptomycin. After washing three times,approximately 100 mg of articular cartilage was aliquoted into micronicstubes and incubated for an additional 24 hours in the above SF media.PRO241 polypeptides were then added at 1% either alone or in combinationwith 18 ng/ml interleukin-1α, a known stimulator of proteoglycan releasefrom cartilage tissue. The supernatant was then harvested and assayedfor the amount of proteoglycans using the 1,9-dimethyl-methylene blue(DMB) colorimetric assay (Farndale and Buttle, Biochem. Biophys. Acta883:173-177 (1985)). A positive result in this assay indicates that thetest polypeptide will find use, for example, in the treatment ofsports-related joint problems, articular cartilage defects,osteoarthritis or rheumatoid arthritis.

When PRO241 polypeptides were tested in the above assay, thepolypeptides demonstrated a marked ability to stimulate release ofproteoglycans from cartilage tissue both basally and after stimulationwith interleukin-1α and at 24 and 72 hours after treatment, therebyindicating that PRO241 polypeptides are useful for stimulatingproteoglycan release from cartilage tissue. As such, PRO241 polypeptidesare useful for the treatment of sports-related joint problems, articularcartilage defects, osteoarthritis or rheumatoid arthritis.

Example 30

In Vitro Antitumor Assay with PRO344 (Assay 161)

The antiproliferative activity of the PRO344 polypeptide was determinedin the investigational, disease-oriented in vitro anti-cancer drugdiscovery assay of the National Cancer Institute (NCI), using asulforhodamine B (SRB) dye binding assay essentially as described bySkehan et al., J. Natl. Cancer Inst. 82:1107-1112 (1990). The 60 tumorcell lines employed in this study (“the NCI panel”), as well asconditions for their maintenance and culture in vitro have beendescribed by Monks et al., J. Natl. Cancer Inst. 83:757-766 (199 1). Thepurpose of this screen is to initially evaluate the cytotoxic and/orcytostatic activity of the test compounds against different types oftumors (Monks et al., supra; Boyd, Cancer: Princ. Pract. Oncol. Update3(10):1-12 [1989]).

Cells from approximately 60 human tumor cell lines were harvested withtrypsin/EDTA (Gibco), washed once, resuspended in IMEM and theirviability was determined. The cell suspensions were added by pipet (100μL volume) into separate 96-well microtiter plates. The cell density forthe 6-day incubation was less than for the 2-day incubation to preventovergrowth. Inoculates were allowed a preincubation period of 24 hoursat 37° C. for stabilization. Dilutions at twice the intended testconcentration were added at time zero in 100 μL aliquots to themicrotiter plate wells (1:2 dilution). Test compounds were evaluated atfive half-log dilutions (1000 to 100,000-fold). Incubations took placefor two days and six days in a 5% CO₂ atmosphere and 100% humidity.

After incubation, the medium was removed and the cells were fixed in 0.1ml of 10% trichloroacetic acid at 40° C. The plates were rinsed fivetimes with deionized water, dried, stained for 30 minutes with 0.1 ml of0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsedfour times with 1% acetic acid to remove unbound dye, dried, and thestain was extracted for five minutes with 0.1 ml of 10 mM Tris base[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) ofsulforhodamine B at 492 rum was measured using a computer-interfaced,96-well microtiter plate reader.

A test sample is considered positive if it shows at least 50% growthinhibitory effect at one or more concentrations. The results are shownin the following Table 23, where the abbreviations are as follows:

NSCL=non-small cell lung carcinoma

CNS=central nervous system

TABLE 23 Tumor Cell Cell Line Test compound Concentration Days Line TypeDesignation PRO344  1.2 nM 2 Leukemia HL-60 (TB) PRO344  1.2 nM 6 RenalUO-31 and CAKI-1 PRO344 14.9 nM 2 Colon KM-12 PRO344 14.9 nM 2 CNSSF-268 PRO344 14.9 nM 2 Ovarian OVCAR-4 PRO344 14.9 nM 2 Renal CAKI-1PRO344 14.9 nM 2 Breast MDA-MB-435 PRO344 14.9 nM 6 Leukemia HL-60 (TB)PRO344 14.9 nM 6 Colon KM-12 PRO344 14.9 nM 6 CNS SF-295 PRO344 14.9 nM6 NSCL HOP62

The results of these assays demonstrate that PRO344 polypeptides areuseful for inhibiting neoplastic growth in a number of different tumorcell types and may be used therapeutically therefor. Antibodies againstPRO344 are useful for affinity purification of this useful polypeptide.Nucleic acids encoding PRO344 polypeptides are useful for therecombinant preparation of these polypeptides.

Example 31

Inhibition of Vascular Endothelial Growth Factor (VEGF) StimulatedProliferationof Endothelial Cell Growth (Assay 9)

The ability of various PRO polypeptides to inhibit VEGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, bovine adrenal cortical capillary endothelial cells (ACE)(from primary culture, maximum of 12-14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and1×penicillin/streptomycin/fungizone. Control wells included thefollowing: (1) no ACE cells added; (2) ACE cells alone; (3) ACE cellsplus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGFplus 5 ng/ml LIF. The test samples, poly-his tagged PRO polypeptides (in100 microliter volumes), were then added to the wells (at dilutions of1%, 0.1% and 0.01%, respectively). The cell cultures were incubated for6-7 days at 37° C./5% CO₂. After the incubation, the media in the wellswas aspirated the cells were washed 1× with PBS. An acid phosphatasereaction mixture (100 microliter; 0.1 M sodium acetate, pH 5.5, 0.1%Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to eachwell. After a 2 hour incubation at 37° C., the reaction was stopped byaddition of 10 microliters 1N NaOH. Optical density (OD) was measured ona microplate reader at 405 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70-90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70-90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

The following polypeptide tested positive in this assay: PRO323.

Example 32

Rod Photoreceptor Cell Survival (Assay 56)

This assay shows that certain polypeptides of the invention act toenhance the survival/proliferation of rod photoreceptor cells and,therefore, are useful for the therapeutic treatment of retinal disordersor injuries including, for example, treating sight loss in mammals dueto retinitis pigmentosum, AMD, etc. Sprague Dawley rat pups at 7 daypostnatal (mixed population: glia and retinal neuronal cell types) arekilled by decapitation following CO₂ anesthesis and the eyes are removedunder sterile conditions. The neural retina is dissected away form thepigment epithelium and other ocular tissue and then dissociated into asingle cell suspension using 0.25% trypsin in Ca²⁺, Mg²⁺-free PBS. Theretinas are incubated at 37° C. for 7-10 minutes after which the trypsinis inactivated by adding 1 ml soybean trypsin inhibitor. The cells areplated at 100,000 cells per well in 96 well plates in DMEM/F12supplemented with N₂. Cells for all experiments are grown at 37° C. in awater saturated atmosphere of 5% CO₂. After 2-3 days in culture, cellsare fixed using 4% paraformaldehyde, and then stained using CellTrackerGreen CMFDA. Rho 4D2 (ascites or IgG 1:100), a monoclonal antibodydirected towards the visual pigment rhodopsin is used to detect rodphotoreceptor cells by indirect immunofluorescence. The results arecalculated as % survival: total number of calcein-rhodopsin positivecells at 2-3 days in culture, divided by the total number of rhodopsinpositive cells at time 2-3 days in culture. The total cells(fluorescent) are quantified at 20× objective magnification using a CCDcamera and NIH image software for MacIntosh. Fields in the well arechosen at random.

The following polypeptides tested positive in this assay: PRO243.

Example 33

Pericyte c-Fos Induction (Assay 93)

This assay shows that certain polypeptides of the invention act toinduce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Specifically, on day 1, pericytes arereceived from VEC Technologies and all but 5 ml of media is removed fromflask. On day 2, the pericytes are trypsinized, washed, spun and thenplated onto 96 well plates. On day 7, the media is removed and thepericytes are treated with 100 μl of PRO polypeptide test samples andcontrols (positive control=DME+5% serum+/−PDGF at 500 ng/ml; negativecontrol=protein 32). Replicates are averaged and SD/CV are determined.Fold increase over Protein 32 (buffer control) value indicated bychemiluminescence units (RLU) luminometer reading verses frequency isplotted on a histogram Two-fold above Protein 32 value is consideredpositive for the assay. ASY Matrix: Growth media =low glucose DMEM=20%FBS+1×pen strep+1×fungizone. Assay Media=low glucose DMEM +5% FBS.

The following polypeptides tested positive in this assay: PRO241.

Example 34

Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay 67)

This example shows that one or more of the polypeptides of the inventionare active as inhibitors of the proliferation of stimulatedT-lymphocytes. Compounds which inhibit proliferation of lymphocytes areuseful therapeutically where suppression of an immune response isbeneficial.

The basic protocol for this assay is described in Current Protocols inImmunology, unit 3.12; edited by J E Coligan, A M Kruisbeek, D HMarglies, E M Shevach, W Strober, National Insitutes of Health,Published by John Wiley & Sons, Inc.

More specifically, in one assay variant, peripheral blood mononuclearcells (PBMC) are isolated from mammalian individuals, for example ahuman volunteer, by leukopheresis (one donor will supply stimulatorPBMCs, the other donor will supply responder PBMCs). If desired, thecells are frozen in fetal bovine serum and DMSO after isolation. Frozencells may be thawed overnight in assay media (37° C., 5% CO₂) and thenwashed and resuspended to 3×10⁶ cells/ml of assay media (RPMI; 10% fetalbovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%non-essential amino acids, 1% pyruvate). The stimulator PBMCs areprepared by irradiating the cells (about 3000 Rads).

The assay is prepared by plating in triplicate wells a mixture of:

100:1 of test sample diluted to 1% or to 0.1%,

50:1 of irradiated stimulator cells, and

50:1 of responder PBMC cells.

100 microliters of cell culture media or 100 microliter of CD4-IgG isused as the control. The wells are then incubated at 37° C., 5% CO₂ for4 days. On day 5, each well is pulsed with tritiated thymidine (1.0mC/well; Amersham). After 6 hours the cells are washed 3 times and thenthe uptake of the label is evaluated.

In another variant of this assay, PBMCs are isolated from the spleens ofBalb/c mice and C57B6 mice. The cells are teased from freshly harvestedspleens in assay media (RPMI; 10% fetal bovine serum, 1%penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential aminoacids, 1% pyruvate) and the PBMCs are isolated by overlaying these cellsover Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20minutes, collecting and washing the mononuclear cell layer in assaymedia and resuspending the cells to 1×10⁷ cells/ml of assay media. Theassay is then conducted as described above.

Any decreases below control is considered to be a positive result for aninhibitory compound, with decreases of less than or equal to 80% beingpreferred. However, any value less than control indicates an inhibitoryeffect for the test protein.

The following polypeptide tested positive in this assay: PRO361.

Example 35

Tissue Expression Distribution

Oligonucleotide probes were constructed from the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO polypeptide-encoding nucleic acid in various different humantissue types provides a diagnostic marker useful for tissue typing, withor without other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. The results of theseassays are shown in Table 24 below.

TABLE 24 Not Significantly Nucleic Acid Significantly Expressed InExpressed In DNA34392-1170 liver, kidney, brain, lung placentaDNA39976-1215 brain lung DNA35595-1228 pancreas, brain, kidney, liverDNA34436-1238 lung, placenta, brain testis DNA44176-1244 liver brain,lung DNA44192-1246 kidney liver DNA44804-1248 lung, brain DNA41234-1242lung, liver, kidney brain DNA45410-1250 lung, brain, kidney, liverDNA46777-1253 liver, placenta, brain

EXAMPLE 36

Fetal Hemoglobin Induction in an Erythroblastic Cell Line (Assay 107)

This assay is useful for screening PRO polypeptides for the ability toinduce the switch from adult hemoglobin to fetal hemoglobin in anerythroblastic cell line. Molecules testing positive in this assay areexpected to be useful for therapeutically treating various mammalianhemoglobin-associated disorders such as the various thalassemias. Theassay is performed as follows. Erythroblastic cells are plated instandard growth medium at 1000 cells/well in a 96 well format. PROpolypeptides are added to the growth medium at a concentration of 0.2%or 2% and the cells are incubated for 5 days at 37° C. As a positivecontrol, cells are treated with 100 μM hemin and as a negative control,the cells are untreated. After 5 days, cell lysates are prepared andanalyzed for the expression of gamma globin (a fetal marker). A positivein the assay is a gamma globin level at least 2-fold above the negativecontrol.

The following polypeptide tested positive in this assay: PRO243.

Example 37

In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [³³-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 10 μ; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the gel was immediately placed on ice. Thewells of gel were flushed, the sample loaded, and run at 180-250 voltsfor 45 minutes. The gel was wrapped in saran wrap and exposed to XARfilm with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper. The tissue was covered with 50 μlof hybridization buffer (3.75 g Dextran Sulfate+6 ml SQ H₂O), vortexedand heated in the microwave for 2 minutes with the cap loosened. Aftercooling on ice, 18.75 ml formamide, 3.75 ml 20×SSC and 9 ml SQ H₂O wereadded, the tissue was vortexed well, and incubated at 42° C. for 14hours.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4 L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4 L).

F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses are as follows.

(1) DNA44804-1248 (PRO357) p15′-GGATTCTAATACGACTCACTATAGGGCTGCCCGCAACCCCTTCAACTG-3′ (SEQ ID NO:111)p2 5′-CTATGAAATTAACCCTCACTAAAGGGACCGCAGCTGGGTGACCGTGTA-3′ (SEQ IDNO:112) (2) DNA52722-1229 (PRO715) p15′-GGATTCTAATACGACTCACTATAGGGCCGCCCCGCCACCTCCT-3′ (SEQ ID NO:113) p25′-CTATGAAATTAACCCTCACTAAAGGGACTCGAGACACCACCTGACCCA3′ (SEQ ID NO:114) p35′-GGATTCTAATACGACTCACTATAGGGCCCAAGGAAGGCAGGAGACTCT-3′ (SEQ ID NO:115)p4 5′-CTATGAAATTAACCCTCACTAAAGGGACTAGGGGGTGGGAATGAAAAG-3′ (SEQ IDNO:116) (3) DNA38113-1230 (PRO327) p15′-GGATTCTAATACGACTCACTATAGGGCCCCCCTGAGCTCTCCCGTGTA-3′ (SEQ ID NO:117)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAAGGCTCGCCACTGGTCGTAGA-3′ (SEQ IDNO:118) (4) DNA35917-1207 (PRO243) p15′-GGATTCTAATACGACTCACTATAGGGCAAGGAGCCGGGACCCAGGAGA-3′ (SEQ ID NO:119)p2 5′-CTATGAAATTAACCCTCACTAAAGGGAGGGGGCCCTTGGTGCTGAGT-3′ (SEQ ID NO:120)

G. Results

In situ analysis was performed on a variety of DNA sequences disclosedherein. The results from these analyses are as follows.

(1) DNA44804-1248 (PRO357)

Low to moderate level expression at sites of bone formation in fetaltissues and in the malignant cells of an osteosarcoma. Possible signalin placenta and cord. All other tissues negative.

Fetal tissues examined (E12-E16 weeks) include: liver, kidney, adrenals,lungs, heart, great vessels, oesophagus, stomach, spleen, gonad, brain,spinal cord and body wall.

Adult human tissues examined: liver, kidney, stomach, spleen, adrenal,pancreas, lung, colonic carcinoma, renal cell carcinoma andosteosarcoma. Acetominophen induced liver injury and hepatic cirrhosis.

Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve, tongue,thymus, adrenal, gastric mucosa and salivary gland.

Rhesus Monkey: cerebrum and cerebellum.

(2) DNA52722-1229 (PRO715)

Generalized high signal seen over many tissues—highest signal seen overplacenta, osteoblasts, injured renal tubules, injured liver, colorectalliver metastasis and gall bladder.

Fetal tissues examined (E12-E16 weeks) include: placenta, umbilicalcord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain,eye, spinal cord, body wall, pelvis and lower limb.

Adult human tissues examined: liver, kidney, adrenal, myocardium, aorta,spleen, lung, skin, chondrosarcoma, eye, stomach, colon, coloniccarcinoma, prostate, bladder mucosa and gall bladder. Acetorninopheninduced liver injury and hepatic cirrhosis.

Rhesus Tissues examined: cerebral cortex (rm), hippocampus (rm)

Chimp Tissues examined: thyroid, parathyroid, lymph node, nerve, tongue,thymus, adrenal, gastric mucosa and salivary gland.

(3) DNA38113-1230 (PRO327)

High level of expression observed in developing mouse and human fetallung. Normal human adult lung, including bronchial epithelium, wasnegative. Expression in submucosa of human fetal trachea, possibly insmooth muscle cells. Expression also observed in non-trophoblastic cellsof uncertain histogenesis in the human placenta. In the mouse expressionwas observed in the developing snout and in the developing tongue. Allother tissues were negative. Speculated function: Probable role inbronchial development.

Fetal tissues examined (E12-E16 weeks) include: placenta, umbilicalcord, liver, kidney, adrenals, thyroid, lungs, heart, great vessels,oesophagus, stomach, small intestine, spleen, thymus, pancreas, brain,eye, spinal cord, body wall, pelvis and lower limb.

Adult tissues examined: liver, kidney, adrenal, myocardium, aorta,spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),hippocampus (rm), cerebellum (rm), penis, eye, bladder, stomach, gastriccarcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid(chimp) ovary (chimp) and chondrosarcoma.

(4) DNA35917-1207 (PRO243)

Cornelia de Lange syndrome (CdLS) is a congenital syndrome. That meansit is present from birth. CdLS is a disorder that causes a delay inphysical, intellectual, and langauge development. The vast majority ofchildren with CdLS are mentally retarded, with the degree of mentalretardation ranging from mild to severe. Reported IQ's from 30 to 85.The average IQ is 53. The head and facial features include small headsize, thin eyebrows which often meet at the midline, long eyelashes,short upturned nose, thin downturned lips, lowset ears and high archedpalate or cleft palate. Other characteristics may include languagedelay, even in the most mildly affected, delayed growth and smallstature, low pitched cry, small hands and feet, incurved fifth fingers,simian creases, and excessive body hair. Diagnosis depends on thepresence of a combination of these characteristics. Many of thesecharacteristics appear in varying degrees. In some cases thesecharacteristics may not be present or be so mild that they will berecognized only when observed by a trained geneticist or other personfamilar with the syndrome. Although much is known about CdLS, recentreports suggest that there is much more to be learned.

In this study additional sections of human fetal face, head, limbs andmouse embryos were examined. No expression was seen in any of the mousetissues. Expression was only seen with the antisense probe.

Expression was observed adjacent to developing limb and facial bones inthe perosteal mesenchyme. The expression was highly specific and wasoften adjacent to areas undergoing vascularization. The distribution isconsistent with the observed skeletal abnormalities in the Cornelia deLange syndrome. Expression was also observed in the developing temporaland occipital lobes of the fetal brain, but was not observed elsewhere.In addition, expression was seen in the ganglia of the developing innerear; the significance of this finding is unclear.

Though these data do not provide functional information, thedistribution is consistent with the sites that are known to be affectedmost severely in this syndrome.

Additionally, faint expression was observed at the cleavage line in thedeveloping synovial joint forming between the femoral head andacetabulum (hip joint). If this pattern of expression were observed atsites of joint formation elsewhere, it might explain the facial and limbabnormalities observed in the Cornelia de Lange syndrome.

Example 38

Activity of PRO243 mRNA in Xenopus Oocytes

In order to demonstrate that the human chordin clone (DNA35917-1207)encoding PRO243 is functional and acts in a manner predicted by theXenopus chordin and Drosophila sog genes, supercoiled plasmid DNA fromDNA35917-1207 was prepared by Qiagen and used for injection into Xenopuslaevis embryos. Micro-injection of Xenopus chordin mRNA intoventrovegetal blastomeres induces secondary (twinned) axes (Sasai etal., Cell 79:779-790 (1994)) and Drosophila sog also induces a secondaryaxis when ectopically expresed on the ventral side of the Xenopus embryo(Holley et al., Nature 376:249-253 (1995) and Schmidt et al.,Development 121:4319-4328 (1995)). The ability of sog to function inXenopus ooctyes suggests that the processes involved in dorsoventralpatterning have been conserved during evolution.

Methods

Manipulation of Xenopus Embryos

Adult female frogs were boosted with 200 I.U. pregnant mare serum 3 daysbefore use and with 800 I.U. of human chorionic gonadotropin the nightbefore injection. Fresh oocytes were squeezed out from female frogs thenext morning and in vitro fertilization of oocytes was performed bymixing oocytes with minced testis from sacrificed male frogs. Developingembryos were maintained and staged according to Nieuwkoop and Faber,Normal Table of Xenopus laevis, N.-H. P. Co., ed. (Amsterdam, 1967).

Fertilized eggs were dejellied with 2% cysteine (pH 7.8) for 10 minutes,washed once with distilled water and transferred to 0.1×MBS with 5%Ficoll. Fertilized eggs were lined on injection trays in 0.1×MBS with 5%Ficoll. Two-cell stage developing Xenopus embryos were injected with 200pg of pRK5 containing wild type chordin (DNA35917-1207) or 200 pg ofpRK5 without an insert as a control. Injected embryos were kept on traysfor another 6 hours, after which they were transferred to 0.1×MBS with50 mg/ml gentamycin until reaching Nieukwkoop stage 37-38.

Results

Injection of human chordin cDNA into single blastomeres resulted in theventralization of the tadpole. The ventralization of the tadpole isvisible in the shortening and kinking of the tail and the expansion ofthe cement gland. The ability of human chordin to function as aventralizing agent in Xenopus shows that the protein encoded byDNA35917-1207 is functional and influences dorsal-ventral patterning infrogs and suggests that the processes involved in dorsoventralpatterning have been conserved during evolution, with mechanisms incommon between humans, flies and frogs.

Deposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC):

Material ATCC Dep. No. Deposit Date DNA34392-1170 ATCC 209526 December10, 1997 DNA35917-1207 ATCC 209508 December 3, 1997 DNA39976-1215 ATCC209524 December 10, 1997 DNA35595-1228 ATCC 209528 December 10, 1997DNA38113-1230 ATCC 209530 December 10, 1997 DNA34436-1238 ATCC 209523December 10, 1997 DNA40592-1242 ATCC 209492 November 21, 1997DNA44176-1244 ATCC 209532 December 10, 1997 DNA44192-1246 ATCC 209531December 10, 1997 DNA39518-1247 ATCC 209529 December 10, 1997DNA44804-1248 ATCC 209527 December 10, 1997 DNA52722-1229 ATCC 209570January 7, 1998 DNA41234-1242 ATCC 209618 February 5, 1998 DNA45410-1250ATCC 209621 February 5, 1998 DNA46777-1253 ATCC 209619 February 5, 1998

These deposit were made under the provisions of Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC § 122 and the Commissioner's rules pursuantthereto (including 37 CFR § 1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 120 <210> SEQ ID NO 1 <211> LENGTH: 2454<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 1ggactaatct gtgggagcag tttattccag tatcacccag ggtgcagcca  #              50caccaggact gtgttgaagg gtgttttttt tcttttaaat gtaatacctc  #             100ctcatctttt cttcttacac agtgtctgag aacatttaca ttatagataa  #             150gtagtacatg gtggataact tctactttta ggaggactac tctcttctga  #             200cagtcctaga ctggtcttct acactaagac accatgaagg agtatgtgct  #             250cctattattc ctggctttgt gctctgccaa acccttcttt agcccttcac  #             300acatcgcact gaagaatatg atgctgaagg atatggaaga cacagatgat  #             350gatgatgatg atgatgatga tgatgatgat gatgaggaca actctctttt  #             400tccaacaaga gagccaagaa gccatttttt tccatttgat ctgtttccaa  #             450tgtgtccatt tggatgtcag tgctattcac gagttgtaca ttgctcagat  #             500ttaggtttga cctcagtccc aaccaacatt ccatttgata ctcgaatgct  #             550tgatcttcaa aacaataaaa ttaaggaaat caaagaaaat gattttaaag  #             600gactcacttc actttatggt ctgatcctga acaacaacaa gctaacgaag  #             650attcacccaa aagcctttct aaccacaaag aagttgcgaa ggctgtatct  #             700gtcccacaat caactaagtg aaataccact taatcttccc aaatcattag  #             750cagaactcag aattcatgaa aataaagtta agaaaataca aaaggacaca  #             800ttcaaaggaa tgaatgcttt acacgttttg gaaatgagtg caaaccctct  #             850tgataataat gggatagagc caggggcatt tgaaggggtg acggtgttcc  #             900atatcagaat tgcagaagca aaactgacct cagttcctaa aggcttacca  #             950ccaactttat tggagcttca cttagattat aataaaattt caacagtgga  #            1000acttgaggat tttaaacgat acaaagaact acaaaggctg ggcctaggaa  #            1050acaacaaaat cacagatatc gaaaatggga gtcttgctaa cataccacgt  #            1100gtgagagaaa tacatttgga aaacaataaa ctaaaaaaaa tcccttcagg  #            1150attaccagag ttgaaatacc tccagataat cttccttcat tctaattcaa  #            1200ttgcaagagt gggagtaaat gacttctgtc caacagtgcc aaagatgaag  #            1250aaatctttat acagtgcaat aagtttattc aacaacccgg tgaaatactg  #            1300ggaaatgcaa cctgcaacat ttcgttgtgt tttgagcaga atgagtgttc  #            1350agcttgggaa ctttggaatg taataattag taattggtaa tgtccattta  #            1400atataagatt caaaaatccc tacatttgga atacttgaac tctattaata  #            1450atggtagtat tatatataca agcaaatatc tattctcaag tggtaagtcc  #            1500actgacttat tttatgacaa gaaatttcaa cggaattttg ccaaactatt  #            1550gatacataag gggttgagag aaacaagcat ctattgcagt ttcctttttg  #            1600cgtacaaatg atcttacata aatctcatgc ttgaccattc ctttcttcat  #            1650aacaaaaaag taagatattc ggtatttaac actttgttat caagcacatt  #            1700ttaaaaagaa ctgtactgta aatggaatgc ttgacttagc aaaatttgtg  #            1750ctctttcatt tgctgttaga aaaacagaat taacaaagac agtaatgtga  #            1800agagtgcatt acactattct tattctttag taacttgggt agtactgtaa  #            1850tatttttaat catcttaaag tatgatttga tataatctta ttgaaattac  #            1900cttatcatgt cttagagccc gtctttatgt ttaaaactaa tttcttaaaa  #            1950taaagccttc agtaaatgtt cattaccaac ttgataaatg ctactcataa  #            2000gagctggttt ggggctatag catatgcttt ttttttttta attattacct  #            2050gatttaaaaa tctctgtaaa aacgtgtagt gtttcataaa atctgtaact  #            2100cgcattttaa tgatccgcta ttataagctt ttaatagcat gaaaattgtt  #            2150aggctatata acattgccac ttcaactcta aggaatattt ttgagatatc  #            2200cctttggaag accttgcttg gaagagcctg gacactaaca attctacacc  #            2250aaattgtctc ttcaaatacg tatggactgg ataactctga gaaacacatc  #            2300tagtataact gaataagcag agcatcaaat taaacagaca gaaaccgaaa  #            2350gctctatata aatgctcaga gttctttatg tatttcttat tggcattcaa  #            2400catatgtaaa atcagaaaac agggaaattt tcattaaaaa tattggtttg  #            2450 aaat                  #                  #                   #           2454 <210> SEQ ID NO 2 <211> LENGTH: 379<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 2Met Lys Glu Tyr Val Leu Leu Leu Phe Leu Al #a Leu Cys Ser Ala  1               5  #                 10  #                 15Lys Pro Phe Phe Ser Pro Ser His Ile Ala Le #u Lys Asn Met Met                 20  #                 25  #                 30Leu Lys Asp Met Glu Asp Thr Asp Asp Asp As #p Asp Asp Asp Asp                 35  #                 40  #                 45Asp Asp Asp Asp Asp Glu Asp Asn Ser Leu Ph #e Pro Thr Arg Glu                 50  #                 55  #                 60Pro Arg Ser His Phe Phe Pro Phe Asp Leu Ph #e Pro Met Cys Pro                 65  #                 70  #                 75Phe Gly Cys Gln Cys Tyr Ser Arg Val Val Hi #s Cys Ser Asp Leu                 80  #                 85  #                 90Gly Leu Thr Ser Val Pro Thr Asn Ile Pro Ph #e Asp Thr Arg Met                 95  #                100  #                105Leu Asp Leu Gln Asn Asn Lys Ile Lys Glu Il #e Lys Glu Asn Asp                110   #               115   #               120Phe Lys Gly Leu Thr Ser Leu Tyr Gly Leu Il #e Leu Asn Asn Asn                125   #               130   #               135Lys Leu Thr Lys Ile His Pro Lys Ala Phe Le #u Thr Thr Lys Lys                140   #               145   #               150Leu Arg Arg Leu Tyr Leu Ser His Asn Gln Le #u Ser Glu Ile Pro                155   #               160   #               165Leu Asn Leu Pro Lys Ser Leu Ala Glu Leu Ar #g Ile His Glu Asn                170   #               175   #               180Lys Val Lys Lys Ile Gln Lys Asp Thr Phe Ly #s Gly Met Asn Ala                185   #               190   #               195Leu His Val Leu Glu Met Ser Ala Asn Pro Le #u Asp Asn Asn Gly                200   #               205   #               210Ile Glu Pro Gly Ala Phe Glu Gly Val Thr Va #l Phe His Ile Arg                215   #               220   #               225Ile Ala Glu Ala Lys Leu Thr Ser Val Pro Ly #s Gly Leu Pro Pro                230   #               235   #               240Thr Leu Leu Glu Leu His Leu Asp Tyr Asn Ly #s Ile Ser Thr Val                245   #               250   #               255Glu Leu Glu Asp Phe Lys Arg Tyr Lys Glu Le #u Gln Arg Leu Gly                260   #               265   #               270Leu Gly Asn Asn Lys Ile Thr Asp Ile Glu As #n Gly Ser Leu Ala                275   #               280   #               285Asn Ile Pro Arg Val Arg Glu Ile His Leu Gl #u Asn Asn Lys Leu                290   #               295   #               300Lys Lys Ile Pro Ser Gly Leu Pro Glu Leu Ly #s Tyr Leu Gln Ile                305   #               310   #               315Ile Phe Leu His Ser Asn Ser Ile Ala Arg Va #l Gly Val Asn Asp                320   #               325   #               330Phe Cys Pro Thr Val Pro Lys Met Lys Lys Se #r Leu Tyr Ser Ala                335   #               340   #               345Ile Ser Leu Phe Asn Asn Pro Val Lys Tyr Tr #p Glu Met Gln Pro                350   #               355   #               360Ala Thr Phe Arg Cys Val Leu Ser Arg Met Se #r Val Gln Leu Gly                365   #               370   #               375Asn Phe Gly Met <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 3 ggaaatgagt gcaaaccctc             #                  #                   # 20 <210> SEQ ID NO 4 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 4 tcccaagctg aacactcatt ctgc          #                   #                24 <210> SEQ ID NO 5<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic Oligonucleotide Pr#obe <400> SEQUENCE: 5gggtgacggt gttccatatc agaattgcag aagcaaaact gacctcagtt  #              50 <210> SEQ ID NO 6 <211> LENGTH: 3441 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 6cggacgcgtg ggcggacgcg tgggcccgcs gcaccgcccc cggcccggcc  #              50ctccgccctc cgcactcgcg cctccctccc tccgcccgct cccgcgccct  #             100cctccctccc tcctccccag ctgtcccgtt cgcgtcatgc cgagcctccc  #             150ggccccgccg gccccgctgc tgctcctcgg gctgctgctg ctcggctccc  #             200ggccggcccg cggcgccggc ccagagcccc ccgtgctgcc catccgttct  #             250gagaaggagc cgctgcccgt tcggggagcg gcaggctgca ccttcggcgg  #             300gaaggtctat gccttggacg agacgtggca cccggaccta gggcagccat  #             350tcggggtgat gcgctgcgtg ctgtgcgcct gcgaggcgcc tcagtggggt  #             400cgccgtacca ggggccctgg cagggtcagc tgcaagaaca tcaaaccaga  #             450gtgcccaacc ccggcctgtg ggcagccgcg ccagctgccg ggacactgct  #             500gccagacctg cccccaggag cgcagcagtt cggagcggca gccgagcggc  #             550ctgtccttcg agtatccgcg ggacccggag catcgcagtt atagcgaccg  #             600cggggagcca ggcgctgagg agcgggcccg tggtgacggc cacacggact  #             650tcgtggcgct gctgacaggg ccgaggtcgc aggcggtggc acgagcccga  #             700gtctcgctgc tgcgctctag cctccgcttc tctatctcct acaggcggct  #             750ggaccgccct accaggatcc gcttctcaga ctccaatggc agtgtcctgt  #             800ttgagcaccc tgcagccccc acccaagatg gcctggtctg tggggtgtgg  #             850cgggcagtgc ctcggttgtc tctgcggctc cttagggcag aacagctgca  #             900tgtggcactt gtgacactca ctcacccttc aggggaggtc tgggggcctc  #             950tcatccggca ccgggccctg gctgcagaga ccttcagtgc catcctgact  #            1000ctagaaggcc ccccacagca gggcgtaggg ggcatcaccc tgctcactct  #            1050cagtgacaca gaggactcct tgcatttttt gctgctcttc cgagggctgc  #            1100tggaacccag gagtggggga ctaacccagg ttcccttgag gctccagatt  #            1150ctacaccagg ggcagctact gcgagaactt caggccaatg tctcagccca  #            1200ggaaccaggc tttgctgagg tgctgcccaa cctgacagtc caggagatgg  #            1250actggctggt gctgggggag ctgcagatgg ccctggagtg ggcaggcagg  #            1300ccagggctgc gcatcagtgg acacattgct gccaggaaga gctgcgacgt  #            1350cctgcaaagt gtcctttgtg gggctgatgc cctgatccca gtccagacgg  #            1400gtgctgccgg ctcagccagc ctcacgctgc taggaaatgg ctccctgatc  #            1450tatcaggtgc aagtggtagg gacaagcagt gaggtggtgg ccatgacact  #            1500ggagaccaag cctcagcgga gggatcagcg cactgtcctg tgccacatgg  #            1550ctggactcca gccaggagga cacacggccg tgggtatctg ccctgggctg  #            1600ggtgcccgag gggctcatat gctgctgcag aatgagctct tcctgaacgt  #            1650gggcaccaag gacttcccag acggagagct tcgggggcac gtggctgccc  #            1700tgccctactg tgggcatagc gcccgccatg acacgctgcc cgtgccccta  #            1750gcaggagccc tggtgctacc ccctgtgaag agccaagcag cagggcacgc  #            1800ctggctttcc ttggataccc actgtcacct gcactatgaa gtgctgctgg  #            1850ctgggcttgg tggctcagaa caaggcactg tcactgccca cctccttggg  #            1900cctcctggaa cgccagggcc tcggcggctg ctgaagggat tctatggctc  #            1950agaggcccag ggtgtggtga aggacctgga gccggaactg ctgcggcacc  #            2000tggcaaaagg catggcctcc ctgatgatca ccaccaaggg tagccccaga  #            2050ggggagctcc gagggcaggt gcacatagcc aaccaatgtg aggttggcgg  #            2100actgcgcctg gaggcggccg gggccgaggg ggtgcgggcg ctgggggctc  #            2150cggatacagc ctctgctgcg ccgcctgtgg tgcctggtct cccggcccta  #            2200gcgcccgcca aacctggtgg tcctgggcgg ccccgagacc ccaacacatg  #            2250cttcttcgag gggcagcagc gcccccacgg ggctcgctgg gcgcccaact  #            2300acgacccgct ctgctcactc tgcacctgcc agagacgaac ggtgatctgt  #            2350gacccggtgg tgtgcccacc gcccagctgc ccacacccgg tgcaggctcc  #            2400cgaccagtgc tgccctgttt gccctgagaa acaagatgtc agagacttgc  #            2450cagggctgcc aaggagccgg gacccaggag agggctgcta ttttgatggt  #            2500gaccggagct ggcgggcagc gggtacgcgg tggcaccccg ttgtgccccc  #            2550ctttggctta attaagtgtg ctgtctgcac ctgcaagggg ggcactggag  #            2600aggtgcactg tgagaaggtg cagtgtcccc ggctggcctg tgcccagcct  #            2650gtgcgtgtca accccaccga ctgctgcaaa cagtgtccag tggggtcggg  #            2700ggcccacccc cagctggggg accccatgca ggctgatggg ccccggggct  #            2750gccgttttgc tgggcagtgg ttcccagaga gtcagagctg gcacccctca  #            2800gtgccccctt ttggagagat gagctgtatc acctgcagat gtggggcagg  #            2850ggtgcctcac tgtgagcggg atgactgttc actgccactg tcctgtggct  #            2900cggggaagga gagtcgatgc tgttcccgct gcacggccca ccggcggccc  #            2950ccagagacca gaactgatcc agagctggag aaagaagccg aaggctctta  #            3000gggagcagcc agagggccaa gtgaccaaga ggatggggcc tgagctgggg  #            3050aaggggtggc atcgaggacc ttcttgcatt ctcctgtggg aagcccagtg  #            3100cctttgctcc tctgtcctgc ctctactccc acccccacta cctctgggaa  #            3150ccacagctcc acaaggggga gaggcagctg ggccagaccg aggtcacagc  #            3200cactccaagt cctgccctgc caccctcggc ctctgtcctg gaagccccac  #            3250ccctttcctc ctgtacataa tgtcactggc ttgttgggat ttttaattta  #            3300tcttcactca gcaccaaggg cccccgacac tccactcctg ctgcccctga  #            3350gctgagcaga gtcattattg gagagttttg tatttattaa aacatttctt  #            3400 tttcagtcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a    #                   # 3441 <210> SEQ ID NO 7 <211> LENGTH: 954<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 7Met Pro Ser Leu Pro Ala Pro Pro Ala Pro Le #u Leu Leu Leu Gly  1               5  #                 10  #                 15Leu Leu Leu Leu Gly Ser Arg Pro Ala Arg Gl #y Ala Gly Pro Glu                 20  #                 25  #                 30Pro Pro Val Leu Pro Ile Arg Ser Glu Lys Gl #u Pro Leu Pro Val                 35  #                 40  #                 45Arg Gly Ala Ala Gly Cys Thr Phe Gly Gly Ly #s Val Tyr Ala Leu                 50  #                 55  #                 60Asp Glu Thr Trp His Pro Asp Leu Gly Gln Pr #o Phe Gly Val Met                 65  #                 70  #                 75Arg Cys Val Leu Cys Ala Cys Glu Ala Pro Gl #n Trp Gly Arg Arg                 80  #                 85  #                 90Thr Arg Gly Pro Gly Arg Val Ser Cys Lys As #n Ile Lys Pro Glu                 95  #                100  #                105Cys Pro Thr Pro Ala Cys Gly Gln Pro Arg Gl #n Leu Pro Gly His                110   #               115   #               120Cys Cys Gln Thr Cys Pro Gln Glu Arg Ser Se #r Ser Glu Arg Gln                125   #               130   #               135Pro Ser Gly Leu Ser Phe Glu Tyr Pro Arg As #p Pro Glu His Arg                140   #               145   #               150Ser Tyr Ser Asp Arg Gly Glu Pro Gly Ala Gl #u Glu Arg Ala Arg                155   #               160   #               165Gly Asp Gly His Thr Asp Phe Val Ala Leu Le #u Thr Gly Pro Arg                170   #               175   #               180Ser Gln Ala Val Ala Arg Ala Arg Val Ser Le #u Leu Arg Ser Ser                185   #               190   #               195Leu Arg Phe Ser Ile Ser Tyr Arg Arg Leu As #p Arg Pro Thr Arg                200   #               205   #               210Ile Arg Phe Ser Asp Ser Asn Gly Ser Val Le #u Phe Glu His Pro                215   #               220   #               225Ala Ala Pro Thr Gln Asp Gly Leu Val Cys Gl #y Val Trp Arg Ala                230   #               235   #               240Val Pro Arg Leu Ser Leu Arg Leu Leu Arg Al #a Glu Gln Leu His                245   #               250   #               255Val Ala Leu Val Thr Leu Thr His Pro Ser Gl #y Glu Val Trp Gly                260   #               265   #               270Pro Leu Ile Arg His Arg Ala Leu Ala Ala Gl #u Thr Phe Ser Ala                275   #               280   #               285Ile Leu Thr Leu Glu Gly Pro Pro Gln Gln Gl #y Val Gly Gly Ile                290   #               295   #               300Thr Leu Leu Thr Leu Ser Asp Thr Glu Asp Se #r Leu His Phe Leu                305   #               310   #               315Leu Leu Phe Arg Gly Leu Leu Glu Pro Arg Se #r Gly Gly Leu Thr                320   #               325   #               330Gln Val Pro Leu Arg Leu Gln Ile Leu His Gl #n Gly Gln Leu Leu                335   #               340   #               345Arg Glu Leu Gln Ala Asn Val Ser Ala Gln Gl #u Pro Gly Phe Ala                350   #               355   #               360Glu Val Leu Pro Asn Leu Thr Val Gln Glu Me #t Asp Trp Leu Val                365   #               370   #               375Leu Gly Glu Leu Gln Met Ala Leu Glu Trp Al #a Gly Arg Pro Gly                380   #               385   #               390Leu Arg Ile Ser Gly His Ile Ala Ala Arg Ly #s Ser Cys Asp Val                395   #               400   #               405Leu Gln Ser Val Leu Cys Gly Ala Asp Ala Le #u Ile Pro Val Gln                410   #               415   #               420Thr Gly Ala Ala Gly Ser Ala Ser Leu Thr Le #u Leu Gly Asn Gly                425   #               430   #               435Ser Leu Ile Tyr Gln Val Gln Val Val Gly Th #r Ser Ser Glu Val                440   #               445   #               450Val Ala Met Thr Leu Glu Thr Lys Pro Gln Ar #g Arg Asp Gln Arg                455   #               460   #               465Thr Val Leu Cys His Met Ala Gly Leu Gln Pr #o Gly Gly His Thr                470   #               475   #               480Ala Val Gly Ile Cys Pro Gly Leu Gly Ala Ar #g Gly Ala His Met                485   #               490   #               495Leu Leu Gln Asn Glu Leu Phe Leu Asn Val Gl #y Thr Lys Asp Phe                500   #               505   #               510Pro Asp Gly Glu Leu Arg Gly His Val Ala Al #a Leu Pro Tyr Cys                515   #               520   #               525Gly His Ser Ala Arg His Asp Thr Leu Pro Va #l Pro Leu Ala Gly                530   #               535   #               540Ala Leu Val Leu Pro Pro Val Lys Ser Gln Al #a Ala Gly His Ala                545   #               550   #               555Trp Leu Ser Leu Asp Thr His Cys His Leu Hi #s Tyr Glu Val Leu                560   #               565   #               570Leu Ala Gly Leu Gly Gly Ser Glu Gln Gly Th #r Val Thr Ala His                575   #               580   #               585Leu Leu Gly Pro Pro Gly Thr Pro Gly Pro Ar #g Arg Leu Leu Lys                590   #               595   #               600Gly Phe Tyr Gly Ser Glu Ala Gln Gly Val Va #l Lys Asp Leu Glu                605   #               610   #               615Pro Glu Leu Leu Arg His Leu Ala Lys Gly Me #t Ala Ser Leu Met                620   #               625   #               630Ile Thr Thr Lys Gly Ser Pro Arg Gly Glu Le #u Arg Gly Gln Val                635   #               640   #               645His Ile Ala Asn Gln Cys Glu Val Gly Gly Le #u Arg Leu Glu Ala                650   #               655   #               660Ala Gly Ala Glu Gly Val Arg Ala Leu Gly Al #a Pro Asp Thr Ala                665   #               670   #               675Ser Ala Ala Pro Pro Val Val Pro Gly Leu Pr #o Ala Leu Ala Pro                680   #               685   #               690Ala Lys Pro Gly Gly Pro Gly Arg Pro Arg As #p Pro Asn Thr Cys                695   #               700   #               705Phe Phe Glu Gly Gln Gln Arg Pro His Gly Al #a Arg Trp Ala Pro                710   #               715   #               720Asn Tyr Asp Pro Leu Cys Ser Leu Cys Thr Cy #s Gln Arg Arg Thr                725   #               730   #               735Val Ile Cys Asp Pro Val Val Cys Pro Pro Pr #o Ser Cys Pro His                740   #               745   #               750Pro Val Gln Ala Pro Asp Gln Cys Cys Pro Va #l Cys Pro Glu Lys                755   #               760   #               765Gln Asp Val Arg Asp Leu Pro Gly Leu Pro Ar #g Ser Arg Asp Pro                770   #               775   #               780Gly Glu Gly Cys Tyr Phe Asp Gly Asp Arg Se #r Trp Arg Ala Ala                785   #               790   #               795Gly Thr Arg Trp His Pro Val Val Pro Pro Ph #e Gly Leu Ile Lys                800   #               805   #               810Cys Ala Val Cys Thr Cys Lys Gly Gly Thr Gl #y Glu Val His Cys                815   #               820   #               825Glu Lys Val Gln Cys Pro Arg Leu Ala Cys Al #a Gln Pro Val Arg                830   #               835   #               840Val Asn Pro Thr Asp Cys Cys Lys Gln Cys Pr #o Val Gly Ser Gly                845   #               850   #               855Ala His Pro Gln Leu Gly Asp Pro Met Gln Al #a Asp Gly Pro Arg                860   #               865   #               870Gly Cys Arg Phe Ala Gly Gln Trp Phe Pro Gl #u Ser Gln Ser Trp                875   #               880   #               885His Pro Ser Val Pro Pro Phe Gly Glu Met Se #r Cys Ile Thr Cys                890   #               895   #               900Arg Cys Gly Ala Gly Val Pro His Cys Glu Ar #g Asp Asp Cys Ser                905   #               910   #               915Leu Pro Leu Ser Cys Gly Ser Gly Lys Glu Se #r Arg Cys Cys Ser                920   #               925   #               930Arg Cys Thr Ala His Arg Arg Pro Pro Glu Th #r Arg Thr Asp Pro                935   #               940   #               945Glu Leu Glu Lys Glu Ala Glu Gly Ser                 950<210> SEQ ID NO 8 <211> LENGTH: 44 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide pr #obe<400> SEQUENCE: 8 gactagttct agatcgcgag cggccgccct tttttttttt tttt   #                   # 44 <210> SEQ ID NO 9 <211> LENGTH: 28<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 9 cggacgcgtg gggcctgcgc acccagct         #                   #             28 <210> SEQ ID NO 10 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 10 gccgctcccc gaacgggcag cggctccttc tcagaa      #                   #       36 <210> SEQ ID NO 11 <211> LENGTH: 36<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 11 ggcgcacagc acgcagcgca tcaccccgaa tggctc      #                   #       36 <210> SEQ ID NO 12 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 12 gtgctgccca tccgttctga gaagga          #                   #              26 <210> SEQ ID NO 13<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 13 gcagggtgct caaacaggac ac           #                   #                 22 <210> SEQ ID NO 14<211> LENGTH: 3231 <212> TYPE: DNA <213> ORGANISM: Homo Sapien<400> SEQUENCE: 14ggcggagcag ccctagccgc caccgtcgct ctcgcagctc tcgtcgccac  #              50tgccaccgcc gccgccgtca ctgcgtcctg gctccggctc ccgcgccctc  #             100ccggccggcc atgcagcccc gccgcgccca ggcgcccggt gcgcagctgc  #             150tgcccgcgct ggccctgctg ctgctgctgc tcggagcggg gccccgaggc  #             200agctccctgg ccaacccggt gcccgccgcg cccttgtctg cgcccgggcc  #             250gtgcgccgcg cagccctgcc ggaatggggg tgtgtgcacc tcgcgccctg  #             300agccggaccc gcagcacccg gcccccgccg gcgagcctgg ctacagctgc  #             350acctgccccg ccgggatctc cggcgccaac tgccagcttg ttgcagatcc  #             400ttgtgccagc aacccttgtc accatggcaa ctgcagcagc agcagcagca  #             450gcagcagcga tggctacctc tgcatttgca atgaaggcta tgaaggtccc  #             500aactgtgaac aggcacttcc cagtctccca gccactggct ggaccgaatc  #             550catggcaccc cgacagcttc agcctgttcc tgctactcag gagcctgaca  #             600aaatcctgcc tcgctctcag gcaacggtga cactgcctac ctggcagccg  #             650aaaacagggc agaaagttgt agaaatgaaa tgggatcaag tggaggtgat  #             700cccagatatt gcctgtggga atgccagttc taacagctct gcgggtggcc  #             750gcctggtatc ctttgaagtg ccacagaaca cctcagtcaa gattcggcaa  #             800gatgccactg cctcactgat tttgctctgg aaggtcacgg ccacaggatt  #             850ccaacagtgc tccctcatag atggacgaag tgtgaccccc cttcaggctt  #             900cagggggact ggtcctcctg gaggagatgc tcgccttggg gaataatcac  #             950tttattggtt ttgtgaatga ttctgtgact aagtctattg tggctttgcg  #            1000cttaactctg gtggtgaagg tcagcacctg tgtgccgggg gagagtcacg  #            1050caaatgactt ggagtgttca ggaaaaggaa aatgcaccac gaagccgtca  #            1100gaggcaactt tttcctgtac ctgtgaggag cagtacgtgg gtactttctg  #            1150tgaagaatac gatgcttgcc agaggaaacc ttgccaaaac aacgcgagct  #            1200gtattgatgc aaatgaaaag caagatggga gcaatttcac ctgtgtttgc  #            1250cttcctggtt atactggaga gctttgccag tccaagattg attactgcat  #            1300cctagaccca tgcagaaatg gagcaacatg catttccagt ctcagtggat  #            1350tcacctgcca gtgtccagaa ggatacttcg gatctgcttg tgaagaaaag  #            1400gtggacccct gcgcctcgtc tccgtgccag aacaacggca cctgctatgt  #            1450ggacggggta cactttacct gcaactgcag cccgggcttc acagggccga  #            1500cctgtgccca gcttattgac ttctgtgccc tcagcccctg tgctcatggc  #            1550acgtgccgca gcgtgggcac cagctacaaa tgcctctgtg atccaggtta  #            1600ccatggcctc tactgtgagg aggaatataa tgagtgcctc tccgctccat  #            1650gcctgaatgc agccacctgc agggacctcg ttaatggcta tgagtgtgtg  #            1700tgcctggcag aatacaaagg aacacactgt gaattgtaca aggatccctg  #            1750cgctaacgtc agctgtctga acggagccac ctgtgacagc gacggcctga  #            1800atggcacgtg catctgtgca cccgggttta caggtgaaga gtgcgacatt  #            1850gacataaatg aatgtgacag taacccctgc caccatggtg ggagctgcct  #            1900ggaccagccc aatggttata actgccactg cccgcatggt tgggtgggag  #            1950caaactgtga gatccacctc caatggaagt ccgggcacat ggcggagagc  #            2000ctcaccaaca tgccacggca ctccctctac atcatcattg gagccctctg  #            2050cgtggccttc atccttatgc tgatcatcct gatcgtgggg atttgccgca  #            2100tcagccgcat tgaataccag ggttcttcca ggccagccta tgaggagttc  #            2150tacaactgcc gcagcatcga cagcgagttc agcaatgcca ttgcatccat  #            2200ccggcatgcc aggtttggaa agaaatcccg gcctgcaatg tatgatgtga  #            2250gccccatcgc ctatgaagat tacagtcctg atgacaaacc cttggtcaca  #            2300ctgattaaaa ctaaagattt gtaatctttt tttggattat ttttcaaaaa  #            2350gatgagatac tacactcatt taaatatttt taagaaaata aaaagcttaa  #            2400gaaatttaaa atgctagctg ctcaagagtt ttcagtagaa tatttaagaa  #            2450ctaattttct gcagctttta gtttggaaaa aatattttaa aaacaaaatt  #            2500tgtgaaacct atagacgatg ttttaatgta ccttcagctc tctaaactgt  #            2550gtgcttctac tagtgtgtgc tcttttcact gtagacacta tcacgagacc  #            2600cagattaatt tctgtggttg ttacagaata agtctaatca aggagaagtt  #            2650tctgtttgac gtttgagtgc cggctttctg agtagagtta ggaaaaccac  #            2700gtaacgtagc atatgatgta taatagagta tacccgttac ttaaaaagaa  #            2750gtctgaaatg ttcgttttgt ggaaaagaaa ctagttaaat ttactattcc  #            2800taacccgaat gaaattagcc tttgccttat tctgtgcatg ggtaagtaac  #            2850ttatttctgc actgttttgt tgaactttgt ggaaacattc tttcgagttt  #            2900gtttttgtca ttttcgtaac agtcgtcgaa ctaggcctca aaaacatacg  #            2950taacgaaaag gcctagcgag gcaaattctg attgatttga atctatattt  #            3000ttctttaaaa agtcaagggt tctatattgt gagtaaatta aatttacatt  #            3050tgagttgttt gttgctaaga ggtagtaaat gtaagagagt actggttcct  #            3100tcagtagtga gtatttctca tagtgcagct ttatttatct ccaggatgtt  #            3150tttgtggctg tatttgattg atatgtgctt cttctgattc ttgctaattt  #            3200 ccaaccatat tgaataaatg tgatcaagtc a        #                   #        3231 <210> SEQ ID NO 15 <211> LENGTH: 737<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 15Met Gln Pro Arg Arg Ala Gln Ala Pro Gly Al #a Gln Leu Leu Pro  1               5  #                 10  #                 15Ala Leu Ala Leu Leu Leu Leu Leu Leu Gly Al #a Gly Pro Arg Gly                 20  #                 25  #                 30Ser Ser Leu Ala Asn Pro Val Pro Ala Ala Pr #o Leu Ser Ala Pro                 35  #                 40  #                 45Gly Pro Cys Ala Ala Gln Pro Cys Arg Asn Gl #y Gly Val Cys Thr                 50  #                 55  #                 60Ser Arg Pro Glu Pro Asp Pro Gln His Pro Al #a Pro Ala Gly Glu                 65  #                 70  #                 75Pro Gly Tyr Ser Cys Thr Cys Pro Ala Gly Il #e Ser Gly Ala Asn                 80  #                 85  #                 90Cys Gln Leu Val Ala Asp Pro Cys Ala Ser As #n Pro Cys His His                 95  #                100  #                105Gly Asn Cys Ser Ser Ser Ser Ser Ser Ser Se #r Asp Gly Tyr Leu                110   #               115   #               120Cys Ile Cys Asn Glu Gly Tyr Glu Gly Pro As #n Cys Glu Gln Ala                125   #               130   #               135Leu Pro Ser Leu Pro Ala Thr Gly Trp Thr Gl #u Ser Met Ala Pro                140   #               145   #               150Arg Gln Leu Gln Pro Val Pro Ala Thr Gln Gl #u Pro Asp Lys Ile                155   #               160   #               165Leu Pro Arg Ser Gln Ala Thr Val Thr Leu Pr #o Thr Trp Gln Pro                170   #               175   #               180Lys Thr Gly Gln Lys Val Val Glu Met Lys Tr #p Asp Gln Val Glu                185   #               190   #               195Val Ile Pro Asp Ile Ala Cys Gly Asn Ala Se #r Ser Asn Ser Ser                200   #               205   #               210Ala Gly Gly Arg Leu Val Ser Phe Glu Val Pr #o Gln Asn Thr Ser                215   #               220   #               225Val Lys Ile Arg Gln Asp Ala Thr Ala Ser Le #u Ile Leu Leu Trp                230   #               235   #               240Lys Val Thr Ala Thr Gly Phe Gln Gln Cys Se #r Leu Ile Asp Gly                245   #               250   #               255Arg Ser Val Thr Pro Leu Gln Ala Ser Gly Gl #y Leu Val Leu Leu                260   #               265   #               270Glu Glu Met Leu Ala Leu Gly Asn Asn His Ph #e Ile Gly Phe Val                275   #               280   #               285Asn Asp Ser Val Thr Lys Ser Ile Val Ala Le #u Arg Leu Thr Leu                290   #               295   #               300Val Val Lys Val Ser Thr Cys Val Pro Gly Gl #u Ser His Ala Asn                305   #               310   #               315Asp Leu Glu Cys Ser Gly Lys Gly Lys Cys Th #r Thr Lys Pro Ser                320   #               325   #               330Glu Ala Thr Phe Ser Cys Thr Cys Glu Glu Gl #n Tyr Val Gly Thr                335   #               340   #               345Phe Cys Glu Glu Tyr Asp Ala Cys Gln Arg Ly #s Pro Cys Gln Asn                350   #               355   #               360Asn Ala Ser Cys Ile Asp Ala Asn Glu Lys Gl #n Asp Gly Ser Asn                365   #               370   #               375Phe Thr Cys Val Cys Leu Pro Gly Tyr Thr Gl #y Glu Leu Cys Gln                380   #               385   #               390Ser Lys Ile Asp Tyr Cys Ile Leu Asp Pro Cy #s Arg Asn Gly Ala                395   #               400   #               405Thr Cys Ile Ser Ser Leu Ser Gly Phe Thr Cy #s Gln Cys Pro Glu                410   #               415   #               420Gly Tyr Phe Gly Ser Ala Cys Glu Glu Lys Va #l Asp Pro Cys Ala                425   #               430   #               435Ser Ser Pro Cys Gln Asn Asn Gly Thr Cys Ty #r Val Asp Gly Val                440   #               445   #               450His Phe Thr Cys Asn Cys Ser Pro Gly Phe Th #r Gly Pro Thr Cys                455   #               460   #               465Ala Gln Leu Ile Asp Phe Cys Ala Leu Ser Pr #o Cys Ala His Gly                470   #               475   #               480Thr Cys Arg Ser Val Gly Thr Ser Tyr Lys Cy #s Leu Cys Asp Pro                485   #               490   #               495Gly Tyr His Gly Leu Tyr Cys Glu Glu Glu Ty #r Asn Glu Cys Leu                500   #               505   #               510Ser Ala Pro Cys Leu Asn Ala Ala Thr Cys Ar #g Asp Leu Val Asn                515   #               520   #               525Gly Tyr Glu Cys Val Cys Leu Ala Glu Tyr Ly #s Gly Thr His Cys                530   #               535   #               540Glu Leu Tyr Lys Asp Pro Cys Ala Asn Val Se #r Cys Leu Asn Gly                545   #               550   #               555Ala Thr Cys Asp Ser Asp Gly Leu Asn Gly Th #r Cys Ile Cys Ala                560   #               565   #               570Pro Gly Phe Thr Gly Glu Glu Cys Asp Ile As #p Ile Asn Glu Cys                575   #               580   #               585Asp Ser Asn Pro Cys His His Gly Gly Ser Cy #s Leu Asp Gln Pro                590   #               595   #               600Asn Gly Tyr Asn Cys His Cys Pro His Gly Tr #p Val Gly Ala Asn                605   #               610   #               615Cys Glu Ile His Leu Gln Trp Lys Ser Gly Hi #s Met Ala Glu Ser                620   #               625   #               630Leu Thr Asn Met Pro Arg His Ser Leu Tyr Il #e Ile Ile Gly Ala                635   #               640   #               645Leu Cys Val Ala Phe Ile Leu Met Leu Ile Il #e Leu Ile Val Gly                650   #               655   #               660Ile Cys Arg Ile Ser Arg Ile Glu Tyr Gln Gl #y Ser Ser Arg Pro                665   #               670   #               675Ala Tyr Glu Glu Phe Tyr Asn Cys Arg Ser Il #e Asp Ser Glu Phe                680   #               685   #               690Ser Asn Ala Ile Ala Ser Ile Arg His Ala Ar #g Phe Gly Lys Lys                695   #               700   #               705Ser Arg Pro Ala Met Tyr Asp Val Ser Pro Il #e Ala Tyr Glu Asp                710   #               715   #               720Tyr Ser Pro Asp Asp Lys Pro Leu Val Thr Le #u Ile Lys Thr Lys                725   #               730   #               735 Asp Leu<210> SEQ ID NO 16 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 16 tgtaaaacga cggccagtta aatagacctg caattattaa tct    #                   # 43 <210> SEQ ID NO 17 <211> LENGTH: 41<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 17 caggaaacag ctatgaccac ctgcacacct gcaaatccat t    #                   #   41 <210> SEQ ID NO 18 <211> LENGTH: 508<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 18ctctggaagg tcacggccac aggattccaa cagtgctccc tcatagatgg  #              50acgaaagtgt gacccccctt tcaggctttc agggggactg gtcctcctgg  #             100aggagatgct cgccttgggg aataatcact ttattggttt tgtgaatgat  #             150tctgtgacta agtctattgt ggctttgcgc ttaactctgg tggtgaaggt  #             200cagcacctgt gtgccggggg agagtcacgc aaatgacttg gagtgttcag  #             250gaaaaggaaa atgcaccacg aagccgtcag aggcaacttt ttcctgtacc  #             300tgtgaggagc agtacgtggg tactttctgt gaagaatacg atgcttgcca  #             350gaggaaacct tgccaaaaca acgcgagctg tattgatgca aatgaaaagc  #             400aagatgggag caatttcacc tgtgtttgcc ttcctggtta tactggagag  #             450ctttgccaac cgaactgaga ttggagcgaa cgacctacac cgaactgaga  #             500 taggggag                 #                  #                   #         508 <210> SEQ ID NO 19 <211> LENGTH: 508<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 19ctctggaagg tcacggccac aggattccaa cagtgctccc tcatagatgg  #              50acgaaagtgt gacccccctt tcaggctttc agggggactg gtcctcctgg  #             100aggagatgct cgccttgggg aataatcact ttattggttt tgtgaatgat  #             150tctgtgacta agtctattgt ggctttgcgc ttaactctgg tggtgaaggt  #             200cagcacctgt gtgccggggg agagtcacgc aaatgacttg gagtgttcag  #             250gaaaaggaaa atgcaccacg aagccgtcag aggcaacttt ttcctgtacc  #             300tgtgaggagc agtacgtggg tactttctgt gaagaatacg atgcttgcca  #             350gaggaaacct tgccaaaaca acgcgagctg tattgatgca aatgaaaagc  #             400aagatgggag caatttcacc tgtgtttgcc ttcctggtta tactggagag  #             450ctttgccaac cgaactgaga ttggagcgaa cgacctacac cgaactgaga  #             500 taggggag                 #                  #                   #         508 <210> SEQ ID NO 20 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 20 ctctggaagg tcacggccac agg           #                   #                23 <210> SEQ ID NO 21<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 21 ctcagttcgg ttggcaaagc tctc          #                   #                24 <210> SEQ ID NO 22<211> LENGTH: 69 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 22cagtgctccc tcatagatgg acgaaagtgt gacccccctt tcaggcgaga  #              50 gctttgccaa ccgaactga              #                  #                   # 69 <210> SEQ ID NO 23 <211> LENGTH: 1520<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 23gctgagtctg ctgctcctgc tgctgctgct ccagcctgta acctgtgcct  #              50acaccacgcc aggccccccc agagccctca ccacgctggg cgcccccaga  #             100gcccacacca tgccgggcac ctacgctccc tcgaccacac tcagtagtcc  #             150cagcacccag ggcctgcaag agcaggcacg ggccctgatg cgggacttcc  #             200cgctcgtgga cggccacaac gacctgcccc tggtcctaag gcaggtttac  #             250cagaaagggc tacaggatgt taacctgcgc aatttcagct acggccagac  #             300cagcctggac aggcttagag atggcctcgt gggcgcccag ttctggtcag  #             350cctatgtgcc atgccagacc caggaccggg atgccctgcg cctcaccctg  #             400gagcagattg acctcatacg ccgcatgtgt gcctcctatt ctgagctgga  #             450gcttgtgacc tcggctaaag ctctgaacga cactcagaaa ttggcctgcc  #             500tcatcggtgt agagggtggc cactcgctgg acaatagcct ctccatctta  #             550cgtaccttct acatgctggg agtgcgctac ctgacgctca cccacacctg  #             600caacacaccc tgggcagaga gctccgctaa gggcgtccac tccttctaca  #             650acaacatcag cgggctgact gactttggtg agaaggtggt ggcagaaatg  #             700aaccgcctgg gcatgatggt agacttatcc catgtctcag atgctgtggc  #             750acggcgggcc ctggaagtgt cacaggcacc tgtgatcttc tcccactcgg  #             800ctgcccgggg tgtgtgcaac agtgctcgga atgttcctga tgacatcctg  #             850cagcttctga agaagaacgg tggcgtcgtg atggtgtctt tgtccatggg  #             900agtaatacag tgcaacccat cagccaatgt gtccactgtg gcagatcact  #             950tcgaccacat caaggctgtc attggatcca agttcatcgg gattggtgga  #            1000gattatgatg gggccggcaa attccctcag gggctggaag acgtgtccac  #            1050atacccggtc ctgatagagg agttgctgag tcgtggctgg agtgaggaag  #            1100agcttcaggg tgtccttcgt ggaaacctgc tgcgggtctt cagacaagtg  #            1150gaaaaggtac aggaagaaaa caaatggcaa agccccttgg aggacaagtt  #            1200cccggatgag cagctgagca gttcctgcca ctccgacctc tcacgtctgc  #            1250gtcagagaca gagtctgact tcaggccagg aactcactga gattcccata  #            1300cactggacag ccaagttacc agccaagtgg tcagtctcag agtcctcccc  #            1350ccacatggcc ccagtccttg cagttgtggc caccttccca gtccttattc  #            1400tgtggctctg atgacccagt tagtcctgcc agatgtcact gtagcaagcc  #            1450acagacaccc cacaaagttc ccctgttgtg caggcacaaa tatttcctga  #            1500 aataaatgtt ttggacatag             #                  #                 152 #0 <210> SEQ ID NO 24 <211> LENGTH: 433<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 24Met Pro Gly Thr Tyr Ala Pro Ser Thr Thr Le #u Ser Ser Pro Ser  1               5  #                 10  #                 15Thr Gln Gly Leu Gln Glu Gln Ala Arg Ala Le #u Met Arg Asp Phe                 20  #                 25  #                 30Pro Leu Val Asp Gly His Asn Asp Leu Pro Le #u Val Leu Arg Gln                 35  #                 40  #                 45Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Le #u Arg Asn Phe Ser                 50  #                 55  #                 60Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg As #p Gly Leu Val Gly                 65  #                 70  #                 75Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gl #n Thr Gln Asp Arg                 80  #                 85  #                 90Asp Ala Leu Arg Leu Thr Leu Glu Gln Ile As #p Leu Ile Arg Arg                 95  #                100  #                105Met Cys Ala Ser Tyr Ser Glu Leu Glu Leu Va #l Thr Ser Ala Lys                110   #               115   #               120Ala Leu Asn Asp Thr Gln Lys Leu Ala Cys Le #u Ile Gly Val Glu                125   #               130   #               135Gly Gly His Ser Leu Asp Asn Ser Leu Ser Il #e Leu Arg Thr Phe                140   #               145   #               150Tyr Met Leu Gly Val Arg Tyr Leu Thr Leu Th #r His Thr Cys Asn                155   #               160   #               165Thr Pro Trp Ala Glu Ser Ser Ala Lys Gly Va #l His Ser Phe Tyr                170   #               175   #               180Asn Asn Ile Ser Gly Leu Thr Asp Phe Gly Gl #u Lys Val Val Ala                185   #               190   #               195Glu Met Asn Arg Leu Gly Met Met Val Asp Le #u Ser His Val Ser                200   #               205   #               210Asp Ala Val Ala Arg Arg Ala Leu Glu Val Se #r Gln Ala Pro Val                215   #               220   #               225Ile Phe Ser His Ser Ala Ala Arg Gly Val Cy #s Asn Ser Ala Arg                230   #               235   #               240Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Ly #s Lys Asn Gly Gly                245   #               250   #               255Val Val Met Val Ser Leu Ser Met Gly Val Il #e Gln Cys Asn Pro                260   #               265   #               270Ser Ala Asn Val Ser Thr Val Ala Asp His Ph #e Asp His Ile Lys                275   #               280   #               285Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gl #y Gly Asp Tyr Asp                290   #               295   #               300Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu As #p Val Ser Thr Tyr                305   #               310   #               315Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gl #y Trp Ser Glu Glu                320   #               325   #               330Glu Leu Gln Gly Val Leu Arg Gly Asn Leu Le #u Arg Val Phe Arg                335   #               340   #               345Gln Val Glu Lys Val Gln Glu Glu Asn Lys Tr #p Gln Ser Pro Leu                350   #               355   #               360Glu Asp Lys Phe Pro Asp Glu Gln Leu Ser Se #r Ser Cys His Ser                365   #               370   #               375Asp Leu Ser Arg Leu Arg Gln Arg Gln Ser Le #u Thr Ser Gly Gln                380   #               385   #               390Glu Leu Thr Glu Ile Pro Ile His Trp Thr Al #a Lys Leu Pro Ala                395   #               400   #               405Lys Trp Ser Val Ser Glu Ser Ser Pro His Me #t Ala Pro Val Leu                410   #               415   #               420Ala Val Val Ala Thr Phe Pro Val Leu Ile Le #u Trp Leu                425   #               430 <210> SEQ ID NO 25<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 25 agttctggtc agcctatgtg cc           #                   #                 22 <210> SEQ ID NO 26<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 26 cgtgatggtg tctttgtcca tggg          #                   #                24 <210> SEQ ID NO 27<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 27 ctccaccaat cccgatgaac ttgg          #                   #                24 <210> SEQ ID NO 28<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 28gagcagattg acctcatacg ccgcatgtgt gcctcctatt ctgagctgga  #              50 <210> SEQ ID NO 29 <211> LENGTH: 1416 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 29aaaacctata aatattccgg attattcata ccgtcccacc atcgggcgcg  #              50gatccgcggc cgcgaattct aaaccaacat gccgggcacc tacgctccct  #             100cgaccacact cagtagtccc agcacccagg gcctgcaaga gcaggcacgg  #             150gccctgatgc gggacttccc gctcgtggac ggccacaacg acctgcccct  #             200ggtcctaagg caggtttacc agaaagggct acaggatgtt aacctgcgca  #             250atttcagcta cggccagacc agcctggaca ggcttagaga tggcctcgtg  #             300ggcgcccagt tctggtcagc ctatgtgcca tgccagaccc aggaccggga  #             350tgccctgcgc ctcaccctgg agcagattga cctcatacgc cgcatgtgtg  #             400cctcctattc tgagctggag cttgtgacct cggctaaagc tctgaacgac  #             450actcagaaat tggcctgcct catcggtgta gagggtggcc actcgctgga  #             500caatagcctc tccatcttac gtaccttcta catgctggga gtgcgctacc  #             550tgacgctcac ccacacctgc aacacaccct gggcagagag ctccgctaag  #             600ggcgtccact ccttctacaa caacatcagc gggctgactg actttggtga  #             650gaaggtggtg gcagaaatga accgcctggg catgatggta gacttatccc  #             700atgtctcaga tgctgtggca cggcgggccc tggaagtgtc acaggcacct  #             750gtgatcttct cccactcggc tgcccggggt gtgtgcaaca gtgctcggaa  #             800tgttcctgat gacatcctgc agcttctgaa gaagaacggt ggcgtcgtga  #             850tggtgtcttt gtccatggga gtaatacagt gcaacccatc agccaatgtg  #             900tccactgtgg cagatcactt cgaccacatc aaggctgtca ttggatccaa  #             950gttcatcggg attggtggag attatgatgg ggccggcaaa ttccctcagg  #            1000ggctggaaga cgtgtccaca tacccggtcc tgatagagga gttgctgagt  #            1050cgtggctgga gtgaggaaga gcttcagggt gtccttcgtg gaaacctgct  #            1100gcgggtcttc agacaagtgg aaaaggtaca ggaagaaaac aaatggcaaa  #            1150gccccttgga ggacaagttc ccggatgagc agctgagcag ttcctgccac  #            1200tccgacctct cacgtctgcg tcagagacag agtctgactt caggccagga  #            1250actcactgag attcccatac actggacagc caagttacca gccaagtggt  #            1300cagtctcaga gtcctccccc caccctgaca aaactcacac atgcccaccg  #            1350tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc  #            1400 aaaacccaag gacacc              #                  #                   #  1416 <210> SEQ ID NO 30 <211> LENGTH: 446<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 30Met Pro Gly Thr Tyr Ala Pro Ser Thr Thr Le #u Ser Ser Pro Ser  1               5  #                 10  #                 15Thr Gln Gly Leu Gln Glu Gln Ala Arg Ala Le #u Met Arg Asp Phe                 20  #                 25  #                 30Pro Leu Val Asp Gly His Asn Asp Leu Pro Le #u Val Leu Arg Gln                 35  #                 40  #                 45Val Tyr Gln Lys Gly Leu Gln Asp Val Asn Le #u Arg Asn Phe Ser                 50  #                 55  #                 60Tyr Gly Gln Thr Ser Leu Asp Arg Leu Arg As #p Gly Leu Val Gly                 65  #                 70  #                 75Ala Gln Phe Trp Ser Ala Tyr Val Pro Cys Gl #n Thr Gln Asp Arg                 80  #                 85  #                 90Asp Ala Leu Arg Leu Thr Leu Glu Gln Ile As #p Leu Ile Arg Arg                 95  #                100  #                105Met Cys Ala Ser Tyr Ser Glu Leu Glu Leu Va #l Thr Ser Ala Lys                110   #               115   #               120Ala Leu Asn Asp Thr Gln Lys Leu Ala Cys Le #u Ile Gly Val Glu                125   #               130   #               135Gly Gly His Ser Leu Asp Asn Ser Leu Ser Il #e Leu Arg Thr Phe                140   #               145   #               150Tyr Met Leu Gly Val Arg Tyr Leu Thr Leu Th #r His Thr Cys Asn                155   #               160   #               165Thr Pro Trp Ala Glu Ser Ser Ala Lys Gly Va #l His Ser Phe Tyr                170   #               175   #               180Asn Asn Ile Ser Gly Leu Thr Asp Phe Gly Gl #u Lys Val Val Ala                185   #               190   #               195Glu Met Asn Arg Leu Gly Met Met Val Asp Le #u Ser His Val Ser                200   #               205   #               210Asp Ala Val Ala Arg Arg Ala Leu Glu Val Se #r Gln Ala Pro Val                215   #               220   #               225Ile Phe Ser His Ser Ala Ala Arg Gly Val Cy #s Asn Ser Ala Arg                230   #               235   #               240Asn Val Pro Asp Asp Ile Leu Gln Leu Leu Ly #s Lys Asn Gly Gly                245   #               250   #               255Val Val Met Val Ser Leu Ser Met Gly Val Il #e Gln Cys Asn Pro                260   #               265   #               270Ser Ala Asn Val Ser Thr Val Ala Asp His Ph #e Asp His Ile Lys                275   #               280   #               285Ala Val Ile Gly Ser Lys Phe Ile Gly Ile Gl #y Gly Asp Tyr Asp                290   #               295   #               300Gly Ala Gly Lys Phe Pro Gln Gly Leu Glu As #p Val Ser Thr Tyr                305   #               310   #               315Pro Val Leu Ile Glu Glu Leu Leu Ser Arg Gl #y Trp Ser Glu Glu                320   #               325   #               330Glu Leu Gln Gly Val Leu Arg Gly Asn Leu Le #u Arg Val Phe Arg                335   #               340   #               345Gln Val Glu Lys Val Gln Glu Glu Asn Lys Tr #p Gln Ser Pro Leu                350   #               355   #               360Glu Asp Lys Phe Pro Asp Glu Gln Leu Ser Se #r Ser Cys His Ser                365   #               370   #               375Asp Leu Ser Arg Leu Arg Gln Arg Gln Ser Le #u Thr Ser Gly Gln                380   #               385   #               390Glu Leu Thr Glu Ile Pro Ile His Trp Thr Al #a Lys Leu Pro Ala                395   #               400   #               405Lys Trp Ser Val Ser Glu Ser Ser Pro His Pr #o Asp Lys Thr His                410   #               415   #               420Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Le #u Gly Gly Pro Ser                425   #               430   #               435Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Th #r                 440  #               445 <210> SEQ ID NO 31 <211> LENGTH: 1790<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 31cgcccagcga cgtgcgggcg gcctggcccg cgccctcccg cgcccggcct  #              50gcgtcccgcg ccctgcgcca ccgccgccga gccgcagccc gccgcgcgcc  #             100cccggcagcg ccggccccat gcccgccggc cgccggggcc ccgccgccca  #             150atccgcgcgg cggccgccgc cgttgctgcc cctgctgctg ctgctctgcg  #             200tcctcggggc gccgcgagcc ggatcaggag cccacacagc tgtgatcagt  #             250ccccaggatc ccacgcttct catcggctcc tccctgctgg ccacctgctc  #             300agtgcacgga gacccaccag gagccaccgc cgagggcctc tactggaccc  #             350tcaacgggcg ccgcctgccc cctgagctct cccgtgtact caacgcctcc  #             400accttggctc tggccctggc caacctcaat gggtccaggc agcggtcggg  #             450ggacaacctc gtgtgccacg cccgtgacgg cagcatcctg gctggctcct  #             500gcctctatgt tggcctgccc ccagagaaac ccgtcaacat cagctgctgg  #             550tccaagaaca tgaaggactt gacctgccgc tggacgccag gggcccacgg  #             600ggagaccttc ctccacacca actactccct caagtacaag cttaggtggt  #             650atggccagga caacacatgt gaggagtacc acacagtggg gccccactcc  #             700tgccacatcc ccaaggacct ggctctcttt acgccctatg agatctgggt  #             750ggaggccacc aaccgcctgg gctctgcccg ctccgatgta ctcacgctgg  #             800atatcctgga tgtggtgacc acggaccccc cgcccgacgt gcacgtgagc  #             850cgcgtcgggg gcctggagga ccagctgagc gtgcgctggg tgtcgccacc  #             900cgccctcaag gatttcctct ttcaagccaa ataccagatc cgctaccgag  #             950tggaggacag tgtggactgg aaggtggtgg acgatgtgag caaccagacc  #            1000tcctgccgcc tggccggcct gaaacccggc accgtgtact tcgtgcaagt  #            1050gcgctgcaac ccctttggca tctatggctc caagaaagcc gggatctgga  #            1100gtgagtggag ccaccccaca gccgcctcca ctccccgcag tgagcgcccg  #            1150ggcccgggcg gcggggcgtg cgaaccgcgg ggcggagagc cgagctcggg  #            1200gccggtgcgg cgcgagctca agcagttcct gggctggctc aagaagcacg  #            1250cgtactgctc caacctcagc ttccgcctct acgaccagtg gcgagcctgg  #            1300atgcagaagt cgcacaagac ccgcaaccag gacgagggga tcctgccctc  #            1350gggcagacgg ggcacggcga gaggtcctgc cagataagct gtaggggctc  #            1400aggccaccct ccctgccacg tggagacgca gaggccgaac ccaaactggg  #            1450gccacctctg taccctcact tcagggcacc tgagccaccc tcagcaggag  #            1500ctggggtggc ccctgagctc caacggccat aacagctctg actcccacgt  #            1550gaggccacct ttgggtgcac cccagtgggt gtgtgtgtgt gtgtgagggt  #            1600tggttgagtt gcctagaacc cctgccaggg ctgggggtga gaaggggagt  #            1650cattactccc cattacctag ggcccctcca aaagagtcct tttaaataaa  #            1700tgagctattt aggtgctgtg attgtgaaaa aaaaaaaaaa aaaaaaaaaa  #            1750 aaaaaaaaaa aaaaaaaaaa aaaaacaaaa aaaaaaaaaa     #                   #  1790 <210> SEQ ID NO 32 <211> LENGTH: 422<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 32Met Pro Ala Gly Arg Arg Gly Pro Ala Ala Gl #n Ser Ala Arg Arg  1               5  #                 10  #                 15Pro Pro Pro Leu Leu Pro Leu Leu Leu Leu Le #u Cys Val Leu Gly                 20  #                 25  #                 30Ala Pro Arg Ala Gly Ser Gly Ala His Thr Al #a Val Ile Ser Pro                 35  #                 40  #                 45Gln Asp Pro Thr Leu Leu Ile Gly Ser Ser Le #u Leu Ala Thr Cys                 50  #                 55  #                 60Ser Val His Gly Asp Pro Pro Gly Ala Thr Al #a Glu Gly Leu Tyr                 65  #                 70  #                 75Trp Thr Leu Asn Gly Arg Arg Leu Pro Pro Gl #u Leu Ser Arg Val                 80  #                 85  #                 90Leu Asn Ala Ser Thr Leu Ala Leu Ala Leu Al #a Asn Leu Asn Gly                 95  #                100  #                105Ser Arg Gln Arg Ser Gly Asp Asn Leu Val Cy #s His Ala Arg Asp                110   #               115   #               120Gly Ser Ile Leu Ala Gly Ser Cys Leu Tyr Va #l Gly Leu Pro Pro                125   #               130   #               135Glu Lys Pro Val Asn Ile Ser Cys Trp Ser Ly #s Asn Met Lys Asp                140   #               145   #               150Leu Thr Cys Arg Trp Thr Pro Gly Ala His Gl #y Glu Thr Phe Leu                155   #               160   #               165His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Ar #g Trp Tyr Gly Gln                170   #               175   #               180Asp Asn Thr Cys Glu Glu Tyr His Thr Val Gl #y Pro His Ser Cys                185   #               190   #               195His Ile Pro Lys Asp Leu Ala Leu Phe Thr Pr #o Tyr Glu Ile Trp                200   #               205   #               210Val Glu Ala Thr Asn Arg Leu Gly Ser Ala Ar #g Ser Asp Val Leu                215   #               220   #               225Thr Leu Asp Ile Leu Asp Val Val Thr Thr As #p Pro Pro Pro Asp                230   #               235   #               240Val His Val Ser Arg Val Gly Gly Leu Glu As #p Gln Leu Ser Val                245   #               250   #               255Arg Trp Val Ser Pro Pro Ala Leu Lys Asp Ph #e Leu Phe Gln Ala                260   #               265   #               270Lys Tyr Gln Ile Arg Tyr Arg Val Glu Asp Se #r Val Asp Trp Lys                275   #               280   #               285Val Val Asp Asp Val Ser Asn Gln Thr Ser Cy #s Arg Leu Ala Gly                290   #               295   #               300Leu Lys Pro Gly Thr Val Tyr Phe Val Gln Va #l Arg Cys Asn Pro                305   #               310   #               315Phe Gly Ile Tyr Gly Ser Lys Lys Ala Gly Il #e Trp Ser Glu Trp                320   #               325   #               330Ser His Pro Thr Ala Ala Ser Thr Pro Arg Se #r Glu Arg Pro Gly                335   #               340   #               345Pro Gly Gly Gly Ala Cys Glu Pro Arg Gly Gl #y Glu Pro Ser Ser                350   #               355   #               360Gly Pro Val Arg Arg Glu Leu Lys Gln Phe Le #u Gly Trp Leu Lys                365   #               370   #               375Lys His Ala Tyr Cys Ser Asn Leu Ser Phe Ar #g Leu Tyr Asp Gln                380   #               385   #               390Trp Arg Ala Trp Met Gln Lys Ser His Lys Th #r Arg Asn Gln Asp                395   #               400   #               405Glu Gly Ile Leu Pro Ser Gly Arg Arg Gly Th #r Ala Arg Gly Pro                410   #               415   #               420 Ala Arg<210> SEQ ID NO 33 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 33 cccgcccgac gtgcacgtga gcc           #                   #                23 <210> SEQ ID NO 34<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 34 tgagccagcc caggaactgc ttg           #                   #                23 <210> SEQ ID NO 35<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 35caagtgcgct gcaacccctt tggcatctat ggctccaaga aagccgggat  #              50 <210> SEQ ID NO 36 <211> LENGTH: 1771 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 36cccacgcgtc cgctggtgtt agatcgagca accctctaaa agcagtttag  #              50agtggtaaaa aaaaaaaaaa acacaccaaa cgctcgcagc cacaaaaggg  #             100atgaaatttc ttctggacat cctcctgctt ctcccgttac tgatcgtctg  #             150ctccctagag tccttcgtga agctttttat tcctaagagg agaaaatcag  #             200tcaccggcga aatcgtgctg attacaggag ctgggcatgg aattgggaga  #             250ctgactgcct atgaatttgc taaacttaaa agcaagctgg ttctctggga  #             300tataaataag catggactgg aggaaacagc tgccaaatgc aagggactgg  #             350gtgccaaggt tcataccttt gtggtagact gcagcaaccg agaagatatt  #             400tacagctctg caaagaaggt gaaggcagaa attggagatg ttagtatttt  #             450agtaaataat gctggtgtag tctatacatc agatttgttt gctacacaag  #             500atcctcagat tgaaaagact tttgaagtta atgtacttgc acatttctgg  #             550actacaaagg catttcttcc tgcaatgacg aagaataacc atggccatat  #             600tgtcactgtg gcttcggcag ctggacatgt ctcggtcccc ttcttactgg  #             650cttactgttc aagcaagttt gctgctgttg gatttcataa aactttgaca  #             700gatgaactgg ctgccttaca aataactgga gtcaaaacaa catgtctgtg  #             750tcctaatttc gtaaacactg gcttcatcaa aaatccaagt acaagtttgg  #             800gacccactct ggaacctgag gaagtggtaa acaggctgat gcatgggatt  #             850ctgactgagc agaagatgat ttttattcca tcttctatag cttttttaac  #             900aacattggaa aggatccttc ctgagcgttt cctggcagtt ttaaaacgaa  #             950aaatcagtgt taagtttgat gcagttattg gatataaaat gaaagcgcaa  #            1000taagcaccta gttttctgaa aactgattta ccaggtttag gttgatgtca  #            1050tctaatagtg ccagaatttt aatgtttgaa cttctgtttt ttctaattat  #            1100ccccatttct tcaatatcat ttttgaggct ttggcagtct tcatttacta  #            1150ccacttgttc tttagccaaa agctgattac atatgatata aacagagaaa  #            1200tacctttaga ggtgacttta aggaaaatga agaaaaagaa ccaaaatgac  #            1250tttattaaaa taatttccaa gattatttgt ggctcacctg aaggctttgc  #            1300aaaatttgta ccataaccgt ttatttaaca tatattttta tttttgattg  #            1350cacttaaatt ttgtataatt tgtgtttctt tttctgttct acataaaatc  #            1400agaaacttca agctctctaa ataaaatgaa ggactatatc tagtggtatt  #            1450tcacaatgaa tatcatgaac tctcaatggg taggtttcat cctacccatt  #            1500gccactctgt ttcctgagag atacctcaca ttccaatgcc aaacatttct  #            1550gcacagggaa gctagaggtg gatacacgtg ttgcaagtat aaaagcatca  #            1600ctgggattta aggagaattg agagaatgta cccacaaatg gcagcaataa  #            1650taaatggatc acacttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  #            1700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  #            1750 aaaaaaaaaa aaaaaaaaaa a            #                  #                1771 <210> SEQ ID NO 37 <211> LENGTH: 300<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 37Met Lys Phe Leu Leu Asp Ile Leu Leu Leu Le #u Pro Leu Leu Ile  1               5  #                 10  #                 15Val Cys Ser Leu Glu Ser Phe Val Lys Leu Ph #e Ile Pro Lys Arg                 20  #                 25  #                 30Arg Lys Ser Val Thr Gly Glu Ile Val Leu Il #e Thr Gly Ala Gly                 35  #                 40  #                 45His Gly Ile Gly Arg Leu Thr Ala Tyr Glu Ph #e Ala Lys Leu Lys                 50  #                 55  #                 60Ser Lys Leu Val Leu Trp Asp Ile Asn Lys Hi #s Gly Leu Glu Glu                 65  #                 70  #                 75Thr Ala Ala Lys Cys Lys Gly Leu Gly Ala Ly #s Val His Thr Phe                 80  #                 85  #                 90Val Val Asp Cys Ser Asn Arg Glu Asp Ile Ty #r Ser Ser Ala Lys                 95  #                100  #                105Lys Val Lys Ala Glu Ile Gly Asp Val Ser Il #e Leu Val Asn Asn                110   #               115   #               120Ala Gly Val Val Tyr Thr Ser Asp Leu Phe Al #a Thr Gln Asp Pro                125   #               130   #               135Gln Ile Glu Lys Thr Phe Glu Val Asn Val Le #u Ala His Phe Trp                140   #               145   #               150Thr Thr Lys Ala Phe Leu Pro Ala Met Thr Ly #s Asn Asn His Gly                155   #               160   #               165His Ile Val Thr Val Ala Ser Ala Ala Gly Hi #s Val Ser Val Pro                170   #               175   #               180Phe Leu Leu Ala Tyr Cys Ser Ser Lys Phe Al #a Ala Val Gly Phe                185   #               190   #               195His Lys Thr Leu Thr Asp Glu Leu Ala Ala Le #u Gln Ile Thr Gly                200   #               205   #               210Val Lys Thr Thr Cys Leu Cys Pro Asn Phe Va #l Asn Thr Gly Phe                215   #               220   #               225Ile Lys Asn Pro Ser Thr Ser Leu Gly Pro Th #r Leu Glu Pro Glu                230   #               235   #               240Glu Val Val Asn Arg Leu Met His Gly Ile Le #u Thr Glu Gln Lys                245   #               250   #               255Met Ile Phe Ile Pro Ser Ser Ile Ala Phe Le #u Thr Thr Leu Glu                260   #               265   #               270Arg Ile Leu Pro Glu Arg Phe Leu Ala Val Le #u Lys Arg Lys Ile                275   #               280   #               285Ser Val Lys Phe Asp Ala Val Ile Gly Tyr Ly #s Met Lys Ala Gln                290   #               295   #               300<210> SEQ ID NO 38 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 38 ggtgaaggca gaaattggag atg           #                   #                23 <210> SEQ ID NO 39<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 39 atcccatgca tcagcctgtt tacc          #                   #                24 <210> SEQ ID NO 40<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 40gctggtgtag tctatacatc agatttgttt gctacacaag atcctcag  #                48 <210> SEQ ID NO 41 <211> LENGTH: 1377<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 41gactagttct cttggagtct gggaggagga aagcggagcc ggcagggagc  #              50gaaccaggac tggggtgacg gcagggcagg gggcgcctgg ccggggagaa  #             100gcgcgggggc tggagcacca ccaactggag ggtccggagt agcgagcgcc  #             150ccgaaggagg ccatcgggga gccgggaggg gggactgcga gaggaccccg  #             200gcgtccgggc tcccggtgcc agcgctatga ggccactcct cgtcctgctg  #             250ctcctgggcc tggcggccgg ctcgccccca ctggacgaca acaagatccc  #             300cagcctctgc ccggggcacc ccggccttcc aggcacgccg ggccaccatg  #             350gcagccaggg cttgccgggc cgcgatggcc gcgacggccg cgacggcgcg  #             400cccggggctc cgggagagaa aggcgagggc gggaggccgg gactgccggg  #             450acctcgaggg gaccccgggc cgcgaggaga ggcgggaccc gcggggccca  #             500ccgggcctgc cggggagtgc tcggtgcctc cgcgatccgc cttcagcgcc  #             550aagcgctccg agagccgggt gcctccgccg tctgacgcac ccttgccctt  #             600cgaccgcgtg ctggtgaacg agcagggaca ttacgacgcc gtcaccggca  #             650agttcacctg ccaggtgcct ggggtctact acttcgccgt ccatgccacc  #             700gtctaccggg ccagcctgca gtttgatctg gtgaagaatg gcgaatccat  #             750tgcctctttc ttccagtttt tcggggggtg gcccaagcca gcctcgctct  #             800cggggggggc catggtgagg ctggagcctg aggaccaagt gtgggtgcag  #             850gtgggtgtgg gtgactacat tggcatctat gccagcatca agacagacag  #             900caccttctcc ggatttctgg tgtactccga ctggcacagc tccccagtct  #             950ttgcttagtg cccactgcaa agtgagctca tgctctcact cctagaagga  #            1000gggtgtgagg ctgacaacca ggtcatccag gagggctggc ccccctggaa  #            1050tattgtgaat gactagggag gtggggtaga gcactctccg tcctgctgct  #            1100ggcaaggaat gggaacagtg gctgtctgcg atcaggtctg gcagcatggg  #            1150gcagtggctg gatttctgcc caagaccaga ggagtgtgct gtgctggcaa  #            1200gtgtaagtcc cccagttgct ctggtccagg agcccacggt ggggtgctct  #            1250cttcctggtc ctctgcttct ctggatcctc cccaccccct cctgctcctg  #            1300gggccggccc ttttctcaga gatcactcaa taaacctaag aaccctcata  #            1350 aaaaaaaaaa aaaaaaaaaa aaaaaaa          #                   #           1377 <210> SEQ ID NO 42<211> LENGTH: 243 <212> TYPE: PRT <213> ORGANISM: Homo Sapien<400> SEQUENCE: 42 Met Arg Pro Leu Leu Val Leu Leu Leu Leu Gl#y Leu Ala Ala Gly   1               5  #                 10 #                 15 Ser Pro Pro Leu Asp Asp Asn Lys Ile Pro Se#r Leu Cys Pro Gly                  20  #                 25 #                 30 His Pro Gly Leu Pro Gly Thr Pro Gly His Hi#s Gly Ser Gln Gly                  35  #                 40 #                 45 Leu Pro Gly Arg Asp Gly Arg Asp Gly Arg As#p Gly Ala Pro Gly                  50  #                 55 #                 60 Ala Pro Gly Glu Lys Gly Glu Gly Gly Arg Pr#o Gly Leu Pro Gly                  65  #                 70 #                 75 Pro Arg Gly Asp Pro Gly Pro Arg Gly Glu Al#a Gly Pro Ala Gly                  80  #                 85 #                 90 Pro Thr Gly Pro Ala Gly Glu Cys Ser Val Pr#o Pro Arg Ser Ala                  95  #                100 #                105 Phe Ser Ala Lys Arg Ser Glu Ser Arg Val Pr#o Pro Pro Ser Asp                 110   #               115  #               120 Ala Pro Leu Pro Phe Asp Arg Val Leu Val As#n Glu Gln Gly His                 125   #               130  #               135 Tyr Asp Ala Val Thr Gly Lys Phe Thr Cys Gl#n Val Pro Gly Val                 140   #               145  #               150 Tyr Tyr Phe Ala Val His Ala Thr Val Tyr Ar#g Ala Ser Leu Gln                 155   #               160  #               165 Phe Asp Leu Val Lys Asn Gly Glu Ser Ile Al#a Ser Phe Phe Gln                 170   #               175  #               180 Phe Phe Gly Gly Trp Pro Lys Pro Ala Ser Le#u Ser Gly Gly Ala                 185   #               190  #               195 Met Val Arg Leu Glu Pro Glu Asp Gln Val Tr#p Val Gln Val Gly                 200   #               205  #               210 Val Gly Asp Tyr Ile Gly Ile Tyr Ala Ser Il#e Lys Thr Asp Ser                 215   #               220  #               225 Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp Tr#p His Ser Ser Pro                 230   #               235  #               240 Val Phe Ala <210> SEQ ID NO 43 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 43 tacaggccca gtcaggacca gggg          #                   #                24 <210> SEQ ID NO 44<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 44 agccagcctc gctctcgg             #                   #                   #  18 <210> SEQ ID NO 45<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 45 gtctgcgatc aggtctgg             #                   #                   #  18 <210> SEQ ID NO 46<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 46 gaaagaggca atggattcgc            #                   #                   # 20 <210> SEQ ID NO 47<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 47 gacttacact tgccagcaca gcac          #                   #                24 <210> SEQ ID NO 48<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 48ggagcaccac caactggagg gtccggagta gcgagcgccc cgaag    #                  #45 <210> SEQ ID NO 49 <211> LENGTH: 1876 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 49ctcttttgtc caccagccca gcctgactcc tggagattgt gaatagctcc  #              50atccagcctg agaaacaagc cgggtggctg agccaggctg tgcacggagc  #             100acctgacggg cccaacagac ccatgctgca tccagagacc tcccctggcc  #             150gggggcatct cctggctgtg ctcctggccc tccttggcac cacctgggca  #             200gaggtgtggc caccccagct gcaggagcag gctccgatgg ccggagccct  #             250gaacaggaag gagagtttct tgctcctctc cctgcacaac cgcctgcgca  #             300gctgggtcca gccccctgcg gctgacatgc ggaggctgga ctggagtgac  #             350agcctggccc aactggctca agccagggca gccctctgtg gaatcccaac  #             400cccgagcctg gcatccggcc tgtggcgcac cctgcaagtg ggctggaaca  #             450tgcagctgct gcccgcgggc ttggcgtcct ttgttgaagt ggtcagccta  #             500tggtttgcag aggggcagcg gtacagccac gcggcaggag agtgtgctcg  #             550caacgccacc tgcacccact acacgcagct cgtgtgggcc acctcaagcc  #             600agctgggctg tgggcggcac ctgtgctctg caggccagac agcgatagaa  #             650gcctttgtct gtgcctactc ccccggaggc aactgggagg tcaacgggaa  #             700gacaatcatc ccctataaga agggtgcctg gtgttcgctc tgcacagcca  #             750gtgtctcagg ctgcttcaaa gcctgggacc atgcaggggg gctctgtgag  #             800gtccccagga atccttgtcg catgagctgc cagaaccatg gacgtctcaa  #             850catcagcacc tgccactgcc actgtccccc tggctacacg ggcagatact  #             900gccaagtgag gtgcagcctg cagtgtgtgc acggccggtt ccgggaggag  #             950gagtgctcgt gcgtctgtga catcggctac gggggagccc agtgtgccac  #            1000caaggtgcat tttcccttcc acacctgtga cctgaggatc gacggagact  #            1050gcttcatggt gtcttcagag gcagacacct attacagagc caggatgaaa  #            1100tgtcagagga aaggcggggt gctggcccag atcaagagcc agaaagtgca  #            1150ggacatcctc gccttctatc tgggccgcct ggagaccacc aacgaggtga  #            1200ctgacagtga cttcgagacc aggaacttct ggatcgggct cacctacaag  #            1250accgccaagg actccttccg ctgggccaca ggggagcacc aggccttcac  #            1300cagttttgcc tttgggcagc ctgacaacca cgggctggtg tggctgagtg  #            1350ctgccatggg gtttggcaac tgcgtggagc tgcaggcttc agctgccttc  #            1400aactggaacg accagcgctg caaaacccga aaccgttaca tctgccagtt  #            1450tgcccaggag cacatctccc ggtggggccc agggtcctga ggcctgacca  #            1500catggctccc tcgcctgccc tgggagcacc ggctctgctt acctgtctgc  #            1550ccacctgtct ggaacaaggg ccaggttaag accacatgcc tcatgtccaa  #            1600agaggtctca gaccttgcac aatgccagaa gttgggcaga gagaggcagg  #            1650gaggccagtg agggccaggg agtgagtgtt agaagaagct ggggcccttc  #            1700gcctgctttt gattgggaag atgggcttca attagatggc gaaggagagg  #            1750acaccgccag tggtccaaaa aggctgctct cttccacctg gcccagaccc  #            1800tgtggggcag cggagcttcc ctgtggcatg aaccccacgg ggtattaaat  #            1850 tatgaatcag ctgaaaaaaa aaaaaa          #                   #            1876 <210> SEQ ID NO 50<211> LENGTH: 455 <212> TYPE: PRT <213> ORGANISM: Homo Sapien<400> SEQUENCE: 50 Met Leu His Pro Glu Thr Ser Pro Gly Arg Gl#y His Leu Leu Ala   1               5  #                 10 #                 15 Val Leu Leu Ala Leu Leu Gly Thr Thr Trp Al#a Glu Val Trp Pro                  20  #                 25 #                 30 Pro Gln Leu Gln Glu Gln Ala Pro Met Ala Gl#y Ala Leu Asn Arg                  35  #                 40 #                 45 Lys Glu Ser Phe Leu Leu Leu Ser Leu His As#n Arg Leu Arg Ser                  50  #                 55 #                 60 Trp Val Gln Pro Pro Ala Ala Asp Met Arg Ar#g Leu Asp Trp Ser                  65  #                 70 #                 75 Asp Ser Leu Ala Gln Leu Ala Gln Ala Arg Al#a Ala Leu Cys Gly                  80  #                 85 #                 90 Ile Pro Thr Pro Ser Leu Ala Ser Gly Leu Tr#p Arg Thr Leu Gln                  95  #                100 #                105 Val Gly Trp Asn Met Gln Leu Leu Pro Ala Gl#y Leu Ala Ser Phe                 110   #               115  #               120 Val Glu Val Val Ser Leu Trp Phe Ala Glu Gl#y Gln Arg Tyr Ser                 125   #               130  #               135 His Ala Ala Gly Glu Cys Ala Arg Asn Ala Th#r Cys Thr His Tyr                 140   #               145  #               150 Thr Gln Leu Val Trp Ala Thr Ser Ser Gln Le#u Gly Cys Gly Arg                 155   #               160  #               165 His Leu Cys Ser Ala Gly Gln Thr Ala Ile Gl#u Ala Phe Val Cys                 170   #               175  #               180 Ala Tyr Ser Pro Gly Gly Asn Trp Glu Val As#n Gly Lys Thr Ile                 185   #               190  #               195 Ile Pro Tyr Lys Lys Gly Ala Trp Cys Ser Le#u Cys Thr Ala Ser                 200   #               205  #               210 Val Ser Gly Cys Phe Lys Ala Trp Asp His Al#a Gly Gly Leu Cys                 215   #               220  #               225 Glu Val Pro Arg Asn Pro Cys Arg Met Ser Cy#s Gln Asn His Gly                 230   #               235  #               240 Arg Leu Asn Ile Ser Thr Cys His Cys His Cy#s Pro Pro Gly Tyr                 245   #               250  #               255 Thr Gly Arg Tyr Cys Gln Val Arg Cys Ser Le#u Gln Cys Val His                 260   #               265  #               270 Gly Arg Phe Arg Glu Glu Glu Cys Ser Cys Va#l Cys Asp Ile Gly                 275   #               280  #               285 Tyr Gly Gly Ala Gln Cys Ala Thr Lys Val Hi#s Phe Pro Phe His                 290   #               295  #               300 Thr Cys Asp Leu Arg Ile Asp Gly Asp Cys Ph#e Met Val Ser Ser                 305   #               310  #               315 Glu Ala Asp Thr Tyr Tyr Arg Ala Arg Met Ly#s Cys Gln Arg Lys                 320   #               325  #               330 Gly Gly Val Leu Ala Gln Ile Lys Ser Gln Ly#s Val Gln Asp Ile                 335   #               340  #               345 Leu Ala Phe Tyr Leu Gly Arg Leu Glu Thr Th#r Asn Glu Val Thr                 350   #               355  #               360 Asp Ser Asp Phe Glu Thr Arg Asn Phe Trp Il#e Gly Leu Thr Tyr                 365   #               370  #               375 Lys Thr Ala Lys Asp Ser Phe Arg Trp Ala Th#r Gly Glu His Gln                 380   #               385  #               390 Ala Phe Thr Ser Phe Ala Phe Gly Gln Pro As#p Asn His Gly Leu                 395   #               400  #               405 Val Trp Leu Ser Ala Ala Met Gly Phe Gly As#n Cys Val Glu Leu                 410   #               415  #               420 Gln Ala Ser Ala Ala Phe Asn Trp Asn Asp Gl#n Arg Cys Lys Thr                 425   #               430  #               435 Arg Asn Arg Tyr Ile Cys Gln Phe Ala Gln Gl#u His Ile Ser Arg                 440   #               445  #               450 Trp Gly Pro Gly Ser                 455<210> SEQ ID NO 51 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 51 aggaacttct ggatcgggct cacc          #                   #                24 <210> SEQ ID NO 52<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 52 gggtctgggc caggtggaag agag          #                   #                24 <210> SEQ ID NO 53<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 53gccaaggact ccttccgctg ggccacaggg gagcaccagg ccttc    #                  #45 <210> SEQ ID NO 54 <211> LENGTH: 2331 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 54cggacgcgtg ggctgggcgc tgcaaagcgt gtcccgccgg gtccccgagc  #              50gtcccgcgcc ctcgccccgc catgctcctg ctgctggggc tgtgcctggg  #             100gctgtccctg tgtgtggggt cgcaggaaga ggcgcagagc tggggccact  #             150cttcggagca ggatggactc agggtcccga ggcaagtcag actgttgcag  #             200aggctgaaaa ccaaaccttt gatgacagaa ttctcagtga agtctaccat  #             250catttcccgt tatgccttca ctacggtttc ctgcagaatg ctgaacagag  #             300cttctgaaga ccaggacatt gagttccaga tgcagattcc agctgcagct  #             350ttcatcacca acttcactat gcttattgga gacaaggtgt atcagggcga  #             400aattacagag agagaaaaga agagtggtga tagggtaaaa gagaaaagga  #             450ataaaaccac agaagaaaat ggagagaagg ggactgaaat attcagagct  #             500tctgcagtga ttcccagcaa ggacaaagcc gcctttttcc tgagttatga  #             550ggagcttctg cagaggcgcc tgggcaagta cgagcacagc atcagcgtgc  #             600ggccccagca gctgtccggg aggctgagcg tggacgtgaa tatcctggag  #             650agcgcgggca tcgcatccct ggaggtgctg ccgcttcaca acagcaggca  #             700gaggggcagt gggcgcgggg aagatgattc tgggcctccc ccatctactg  #             750tcattaacca aaatgaaaca tttgccaaca taatttttaa acctactgta  #             800gtacaacaag ccaggattgc ccagaatgga attttgggag actttatcat  #             850tagatatgac gtcaatagag aacagagcat tggggacatc caggttctaa  #             900atggctattt tgtgcactac tttgctccta aagaccttcc tcctttaccc  #             950aagaatgtgg tattcgtgct tgacagcagt gcttctatgg tgggaaccaa  #            1000actccggcag accaaggatg ccctcttcac aattctccat gacctccgac  #            1050cccaggaccg tttcagtatc attggatttt ccaaccggat caaagtatgg  #            1100aaggaccact tgatatcagt cactccagac agcatcaggg atgggaaagt  #            1150gtacattcac catatgtcac ccactggagg cacagacatc aacggggccc  #            1200tgcagagggc catcaggctc ctcaacaagt acgtggccca cagtggcatt  #            1250ggagaccgga gcgtgtccct catcgtcttc ctgacggatg ggaagcccac  #            1300ggtcggggag acgcacaccc tcaagatcct caacaacacc cgagaggccg  #            1350cccgaggcca agtctgcatc ttcaccattg gcatcggcaa cgacgtggac  #            1400ttcaggctgc tggagaaact gtcgctggag aactgtggcc tcacacggcg  #            1450cgtgcacgag gaggaggacg caggctcgca gctcatcggg ttctacgatg  #            1500aaatcaggac cccgctcctc tctgacatcc gcatcgatta tccccccagc  #            1550tcagtggtgc aggccaccaa gaccctgttc cccaactact tcaacggctc  #            1600ggagatcatc attgcgggga agctggtgga caggaagctg gatcacctgc  #            1650acgtggaggt caccgccagc aacagtaaga aattcatcat cctgaagaca  #            1700gatgtgcctg tgcggcctca gaaggcaggg aaagatgtca caggaagccc  #            1750caggcctgga ggcgatggag agggggacac caaccacatc gagcgtctct  #            1800ggagctacct caccacaaag gagctgctga gctcctggct gcaaagtgac  #            1850gatgaaccgg agaaggagcg gctgcggcag cgggcccagg ccctggctgt  #            1900gagctaccgc ttcctcactc ccttcacctc catgaagctg agggggccgg  #            1950tcccacgcat ggatggcctg gaggaggccc acggcatgtc ggctgccatg  #            2000ggacccgaac cggtggtgca gagcgtgcga ggagctggca cgcagccagg  #            2050acctttgctc aagaagccaa actccgtcaa aaaaaaacaa aacaaaacaa  #            2100aaaaaagaca tgggagagat ggtgtttttc ctctccacca cctggggata  #            2150cgatgagaag atggccacct gcaagccagg aagacggccc tcaccagaca  #            2200ccatgtctgc tggcaccttg atcttggacc tcccagcctc cagaactgtg  #            2250agaaataaat gtgttttgtt taagctaaaa aaaaaaaaaa aaaaaaaaaa  #            2300 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a        #                   #        2331 <210> SEQ ID NO 55 <211> LENGTH: 694<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 55Met Leu Leu Leu Leu Gly Leu Cys Leu Gly Le #u Ser Leu Cys Val  1               5  #                 10  #                 15Gly Ser Gln Glu Glu Ala Gln Ser Trp Gly Hi #s Ser Ser Glu Gln                 20  #                 25  #                 30Asp Gly Leu Arg Val Pro Arg Gln Val Arg Le #u Leu Gln Arg Leu                 35  #                 40  #                 45Lys Thr Lys Pro Leu Met Thr Glu Phe Ser Va #l Lys Ser Thr Ile                 50  #                 55  #                 60Ile Ser Arg Tyr Ala Phe Thr Thr Val Ser Cy #s Arg Met Leu Asn                 65  #                 70  #                 75Arg Ala Ser Glu Asp Gln Asp Ile Glu Phe Gl #n Met Gln Ile Pro                 80  #                 85  #                 90Ala Ala Ala Phe Ile Thr Asn Phe Thr Met Le #u Ile Gly Asp Lys                 95  #                100  #                105Val Tyr Gln Gly Glu Ile Thr Glu Arg Glu Ly #s Lys Ser Gly Asp                110   #               115   #               120Arg Val Lys Glu Lys Arg Asn Lys Thr Thr Gl #u Glu Asn Gly Glu                125   #               130   #               135Lys Gly Thr Glu Ile Phe Arg Ala Ser Ala Va #l Ile Pro Ser Lys                140   #               145   #               150Asp Lys Ala Ala Phe Phe Leu Ser Tyr Glu Gl #u Leu Leu Gln Arg                155   #               160   #               165Arg Leu Gly Lys Tyr Glu His Ser Ile Ser Va #l Arg Pro Gln Gln                170   #               175   #               180Leu Ser Gly Arg Leu Ser Val Asp Val Asn Il #e Leu Glu Ser Ala                185   #               190   #               195Gly Ile Ala Ser Leu Glu Val Leu Pro Leu Hi #s Asn Ser Arg Gln                200   #               205   #               210Arg Gly Ser Gly Arg Gly Glu Asp Asp Ser Gl #y Pro Pro Pro Ser                215   #               220   #               225Thr Val Ile Asn Gln Asn Glu Thr Phe Ala As #n Ile Ile Phe Lys                230   #               235   #               240Pro Thr Val Val Gln Gln Ala Arg Ile Ala Gl #n Asn Gly Ile Leu                245   #               250   #               255Gly Asp Phe Ile Ile Arg Tyr Asp Val Asn Ar #g Glu Gln Ser Ile                260   #               265   #               270Gly Asp Ile Gln Val Leu Asn Gly Tyr Phe Va #l His Tyr Phe Ala                275   #               280   #               285Pro Lys Asp Leu Pro Pro Leu Pro Lys Asn Va #l Val Phe Val Leu                290   #               295   #               300Asp Ser Ser Ala Ser Met Val Gly Thr Lys Le #u Arg Gln Thr Lys                305   #               310   #               315Asp Ala Leu Phe Thr Ile Leu His Asp Leu Ar #g Pro Gln Asp Arg                320   #               325   #               330Phe Ser Ile Ile Gly Phe Ser Asn Arg Ile Ly #s Val Trp Lys Asp                335   #               340   #               345His Leu Ile Ser Val Thr Pro Asp Ser Ile Ar #g Asp Gly Lys Val                350   #               355   #               360Tyr Ile His His Met Ser Pro Thr Gly Gly Th #r Asp Ile Asn Gly                365   #               370   #               375Ala Leu Gln Arg Ala Ile Arg Leu Leu Asn Ly #s Tyr Val Ala His                380   #               385   #               390Ser Gly Ile Gly Asp Arg Ser Val Ser Leu Il #e Val Phe Leu Thr                395   #               400   #               405Asp Gly Lys Pro Thr Val Gly Glu Thr His Th #r Leu Lys Ile Leu                410   #               415   #               420Asn Asn Thr Arg Glu Ala Ala Arg Gly Gln Va #l Cys Ile Phe Thr                425   #               430   #               435Ile Gly Ile Gly Asn Asp Val Asp Phe Arg Le #u Leu Glu Lys Leu                440   #               445   #               450Ser Leu Glu Asn Cys Gly Leu Thr Arg Arg Va #l His Glu Glu Glu                455   #               460   #               465Asp Ala Gly Ser Gln Leu Ile Gly Phe Tyr As #p Glu Ile Arg Thr                470   #               475   #               480Pro Leu Leu Ser Asp Ile Arg Ile Asp Tyr Pr #o Pro Ser Ser Val                485   #               490   #               495Val Gln Ala Thr Lys Thr Leu Phe Pro Asn Ty #r Phe Asn Gly Ser                500   #               505   #               510Glu Ile Ile Ile Ala Gly Lys Leu Val Asp Ar #g Lys Leu Asp His                515   #               520   #               525Leu His Val Glu Val Thr Ala Ser Asn Ser Ly #s Lys Phe Ile Ile                530   #               535   #               540Leu Lys Thr Asp Val Pro Val Arg Pro Gln Ly #s Ala Gly Lys Asp                545   #               550   #               555Val Thr Gly Ser Pro Arg Pro Gly Gly Asp Gl #y Glu Gly Asp Thr                560   #               565   #               570Asn His Ile Glu Arg Leu Trp Ser Tyr Leu Th #r Thr Lys Glu Leu                575   #               580   #               585Leu Ser Ser Trp Leu Gln Ser Asp Asp Glu Pr #o Glu Lys Glu Arg                590   #               595   #               600Leu Arg Gln Arg Ala Gln Ala Leu Ala Val Se #r Tyr Arg Phe Leu                605   #               610   #               615Thr Pro Phe Thr Ser Met Lys Leu Arg Gly Pr #o Val Pro Arg Met                620   #               625   #               630Asp Gly Leu Glu Glu Ala His Gly Met Ser Al #a Ala Met Gly Pro                635   #               640   #               645Glu Pro Val Val Gln Ser Val Arg Gly Ala Gl #y Thr Gln Pro Gly                650   #               655   #               660Pro Leu Leu Lys Lys Pro Asn Ser Val Lys Ly #s Lys Gln Asn Lys                665   #               670   #               675Thr Lys Lys Arg His Gly Arg Asp Gly Val Ph #e Pro Leu His His                680   #               685   #               690Leu Gly Ile Arg <210> SEQ ID NO 56 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 56 gtgggaacca aactccggca gacc          #                   #                24 <210> SEQ ID NO 57<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 57 cacatcgagc gtctctgg             #                   #                   #  18 <210> SEQ ID NO 58<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 58 agccgctcct tctccggttc atcg          #                   #                24 <210> SEQ ID NO 59<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 59tggaaggacc acttgatatc agtcactcca gacagcatca gggatggg  #                48 <210> SEQ ID NO 60 <211> LENGTH: 1413<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 60cggacgcgtg gggtgcccga catggcgagt gtagtgctgc cgagcggatc  #              50ccagtgtgcg gcggcagcgg cggcggcggc gcctcccggg ctccggcttc  #             100tgctgttgct cttctccgcc gcggcactga tccccacagg tgatgggcag  #             150aatctgttta cgaaagacgt gacagtgatc gagggagagg ttgcgaccat  #             200cagttgccaa gtcaataaga gtgacgactc tgtgattcag ctactgaatc  #             250ccaacaggca gaccatttat ttcagggact tcaggccttt gaaggacagc  #             300aggtttcagt tgctgaattt ttctagcagt gaactcaaag tatcattgac  #             350aaacgtctca atttctgatg aaggaagata cttttgccag ctctataccg  #             400atcccccaca ggaaagttac accaccatca cagtcctggt cccaccacgt  #             450aatctgatga tcgatatcca gaaagacact gcggtggaag gtgaggagat  #             500tgaagtcaac tgcactgcta tggccagcaa gccagccacg actatcaggt  #             550ggttcaaagg gaacacagag ctaaaaggca aatcggaggt ggaagagtgg  #             600tcagacatgt acactgtgac cagtcagctg atgctgaagg tgcacaagga  #             650ggacgatggg gtcccagtga tctgccaggt ggagcaccct gcggtcactg  #             700gaaacctgca gacccagcgg tatctagaag tacagtataa gcctcaagtg  #             750cacattcaga tgacttatcc tctacaaggc ttaacccggg aaggggacgc  #             800gcttgagtta acatgtgaag ccatcgggaa gccccagcct gtgatggtaa  #             850cttgggtgag agtcgatgat gaaatgcctc aacacgccgt actgtctggg  #             900cccaacctgt tcatcaataa cctaaacaaa acagataatg gtacataccg  #             950ctgtgaagct tcaaacatag tggggaaagc tcactcggat tatatgctgt  #            1000atgtatacga tccccccaca actatccctc ctcccacaac aaccaccacc  #            1050accaccacca ccaccaccac caccatcctt accatcatca cagattcccg  #            1100agcaggtgaa gaaggctcga tcagggcagt ggatcatgcc gtgatcggtg  #            1150gcgtcgtggc ggtggtggtg ttcgccatgc tgtgcttgct catcattctg  #            1200gggcgctatt ttgccagaca taaaggtaca tacttcactc atgaagccaa  #            1250aggagccgat gacgcagcag acgcagacac agctataatc aatgcagaag  #            1300gaggacagaa caactccgaa gaaaagaaag agtacttcat ctagatcagc  #            1350ctttttgttt caatgaggtg tccaactggc cctatttaga tgataaagag  #            1400 acagtgatat tgg               #                  #                   #    1413 <210> SEQ ID NO 61 <211> LENGTH: 440<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 61Met Ala Ser Val Val Leu Pro Ser Gly Ser Gl #n Cys Ala Ala Ala  1               5  #                 10  #                 15Ala Ala Ala Ala Ala Pro Pro Gly Leu Arg Le #u Leu Leu Leu Leu                 20  #                 25  #                 30Phe Ser Ala Ala Ala Leu Ile Pro Thr Gly As #p Gly Gln Asn Leu                 35  #                 40  #                 45Phe Thr Lys Asp Val Thr Val Ile Glu Gly Gl #u Val Ala Thr Ile                 50  #                 55  #                 60Ser Cys Gln Val Asn Lys Ser Asp Asp Ser Va #l Ile Gln Leu Leu                 65  #                 70  #                 75Asn Pro Asn Arg Gln Thr Ile Tyr Phe Arg As #p Phe Arg Pro Leu                 80  #                 85  #                 90Lys Asp Ser Arg Phe Gln Leu Leu Asn Phe Se #r Ser Ser Glu Leu                 95  #                100  #                105Lys Val Ser Leu Thr Asn Val Ser Ile Ser As #p Glu Gly Arg Tyr                110   #               115   #               120Phe Cys Gln Leu Tyr Thr Asp Pro Pro Gln Gl #u Ser Tyr Thr Thr                125   #               130   #               135Ile Thr Val Leu Val Pro Pro Arg Asn Leu Me #t Ile Asp Ile Gln                140   #               145   #               150Lys Asp Thr Ala Val Glu Gly Glu Glu Ile Gl #u Val Asn Cys Thr                155   #               160   #               165Ala Met Ala Ser Lys Pro Ala Thr Thr Ile Ar #g Trp Phe Lys Gly                170   #               175   #               180Asn Thr Glu Leu Lys Gly Lys Ser Glu Val Gl #u Glu Trp Ser Asp                185   #               190   #               195Met Tyr Thr Val Thr Ser Gln Leu Met Leu Ly #s Val His Lys Glu                200   #               205   #               210Asp Asp Gly Val Pro Val Ile Cys Gln Val Gl #u His Pro Ala Val                215   #               220   #               225Thr Gly Asn Leu Gln Thr Gln Arg Tyr Leu Gl #u Val Gln Tyr Lys                230   #               235   #               240Pro Gln Val His Ile Gln Met Thr Tyr Pro Le #u Gln Gly Leu Thr                245   #               250   #               255Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys Gl #u Ala Ile Gly Lys                260   #               265   #               270Pro Gln Pro Val Met Val Thr Trp Val Arg Va #l Asp Asp Glu Met                275   #               280   #               285Pro Gln His Ala Val Leu Ser Gly Pro Asn Le #u Phe Ile Asn Asn                290   #               295   #               300Leu Asn Lys Thr Asp Asn Gly Thr Tyr Arg Cy #s Glu Ala Ser Asn                305   #               310   #               315Ile Val Gly Lys Ala His Ser Asp Tyr Met Le #u Tyr Val Tyr Asp                320   #               325   #               330Pro Pro Thr Thr Ile Pro Pro Pro Thr Thr Th #r Thr Thr Thr Thr                335   #               340   #               345Thr Thr Thr Thr Thr Thr Ile Leu Thr Ile Il #e Thr Asp Ser Arg                350   #               355   #               360Ala Gly Glu Glu Gly Ser Ile Arg Ala Val As #p His Ala Val Ile                365   #               370   #               375Gly Gly Val Val Ala Val Val Val Phe Ala Me #t Leu Cys Leu Leu                380   #               385   #               390Ile Ile Leu Gly Arg Tyr Phe Ala Arg His Ly #s Gly Thr Tyr Phe                395   #               400   #               405Thr His Glu Ala Lys Gly Ala Asp Asp Ala Al #a Asp Ala Asp Thr                410   #               415   #               420Ala Ile Ile Asn Ala Glu Gly Gly Gln Asn As #n Ser Glu Glu Lys                425   #               430   #               435Lys Glu Tyr Phe Ile                 440 <210> SEQ ID NO 62<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 62 ggcttctgct gttgctcttc tccg          #                   #                24 <210> SEQ ID NO 63<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 63 gtacactgtg accagtcagc            #                   #                   # 20 <210> SEQ ID NO 64<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 64 atcatcacag attcccgagc            #                   #                   # 20 <210> SEQ ID NO 65<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 65 ttcaatctcc tcaccttcca ccgc          #                   #                24 <210> SEQ ID NO 66<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 66 atagctgtgt ctgcgtctgc tgcg          #                   #                24 <210> SEQ ID NO 67<211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 67cgcggcactg atccccacag gtgatgggca gaatctgttt acgaaagacg  #              50 <210> SEQ ID NO 68 <211> LENGTH: 2555 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 68ggggcgggtg gacgcggact cgaacgcagt tgcttcggga cccaggaccc  #              50cctcgggccc gacccgccag gaaagactga ggccgcggcc tgccccgccc  #             100ggctccctgc gccgccgccg cctcccggga cagaagatgt gctccagggt  #             150ccctctgctg ctgccgctgc tcctgctact ggccctgggg cctggggtgc  #             200agggctgccc atccggctgc cagtgcagcc agccacagac agtcttctgc  #             250actgcccgcc aggggaccac ggtgccccga gacgtgccac ccgacacggt  #             300ggggctgtac gtctttgaga acggcatcac catgctcgac gcaagcagct  #             350ttgccggcct gccgggcctg cagctcctgg acctgtcaca gaaccagatc  #             400gccagcctgc gcctgccccg cctgctgctg ctggacctca gccacaacag  #             450cctcctggcc ctggagcccg gcatcctgga cactgccaac gtggaggcgc  #             500tgcggctggc tggtctgggg ctgcagcagc tggacgaggg gctcttcagc  #             550cgcttgcgca acctccacga cctggatgtg tccgacaacc agctggagcg  #             600agtgccacct gtgatccgag gcctccgggg cctgacgcgc ctgcggctgg  #             650ccggcaacac ccgcattgcc cagctgcggc ccgaggacct ggccggcctg  #             700gctgccctgc aggagctgga tgtgagcaac ctaagcctgc aggccctgcc  #             750tggcgacctc tcgggcctct tcccccgcct gcggctgctg gcagctgccc  #             800gcaacccctt caactgcgtg tgccccctga gctggtttgg cccctgggtg  #             850cgcgagagcc acgtcacact ggccagccct gaggagacgc gctgccactt  #             900cccgcccaag aacgctggcc ggctgctcct ggagcttgac tacgccgact  #             950ttggctgccc agccaccacc accacagcca cagtgcccac cacgaggccc  #            1000gtggtgcggg agcccacagc cttgtcttct agcttggctc ctacctggct  #            1050tagccccaca gcgccggcca ctgaggcccc cagcccgccc tccactgccc  #            1100caccgactgt agggcctgtc ccccagcccc aggactgccc accgtccacc  #            1150tgcctcaatg ggggcacatg ccacctgggg acacggcacc acctggcgtg  #            1200cttgtgcccc gaaggcttca cgggcctgta ctgtgagagc cagatggggc  #            1250aggggacacg gcccagccct acaccagtca cgccgaggcc accacggtcc  #            1300ctgaccctgg gcatcgagcc ggtgagcccc acctccctgc gcgtggggct  #            1350gcagcgctac ctccagggga gctccgtgca gctcaggagc ctccgtctca  #            1400cctatcgcaa cctatcgggc cctgataagc ggctggtgac gctgcgactg  #            1450cctgcctcgc tcgctgagta cacggtcacc cagctgcggc ccaacgccac  #            1500ttactccgtc tgtgtcatgc ctttggggcc cgggcgggtg ccggagggcg  #            1550aggaggcctg cggggaggcc catacacccc cagccgtcca ctccaaccac  #            1600gccccagtca cccaggcccg cgagggcaac ctgccgctcc tcattgcgcc  #            1650cgccctggcc gcggtgctcc tggccgcgct ggctgcggtg ggggcagcct  #            1700actgtgtgcg gcgggggcgg gccatggcag cagcggctca ggacaaaggg  #            1750caggtggggc caggggctgg gcccctggaa ctggagggag tgaaggtccc  #            1800cttggagcca ggcccgaagg caacagaggg cggtggagag gccctgccca  #            1850gcgggtctga gtgtgaggtg ccactcatgg gcttcccagg gcctggcctc  #            1900cagtcacccc tccacgcaaa gccctacatc taagccagag agagacaggg  #            1950cagctggggc cgggctctca gccagtgaga tggccagccc cctcctgctg  #            2000ccacaccacg taagttctca gtcccaacct cggggatgtg tgcagacagg  #            2050gctgtgtgac cacagctggg ccctgttccc tctggacctc ggtctcctca  #            2100tctgtgagat gctgtggccc agctgacgag ccctaacgtc cccagaaccg  #            2150agtgcctatg aggacagtgt ccgccctgcc ctccgcaacg tgcagtccct  #            2200gggcacggcg ggccctgcca tgtgctggta acgcatgcct gggccctgct  #            2250gggctctccc actccaggcg gaccctgggg gccagtgaag gaagctcccg  #            2300gaaagagcag agggagagcg ggtaggcggc tgtgtgactc tagtcttggc  #            2350cccaggaagc gaaggaacaa aagaaactgg aaaggaagat gctttaggaa  #            2400catgttttgc ttttttaaaa tatatatata tttataagag atcctttccc  #            2450atttattctg ggaagatgtt tttcaaactc agagacaagg actttggttt  #            2500ttgtaagaca aacgatgata tgaaggcctt ttgtaagaaa aaataaaaaa  #            2550 aaaaa                  #                  #                   #          2555 <210> SEQ ID NO 69 <211> LENGTH: 598<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 69Met Cys Ser Arg Val Pro Leu Leu Leu Pro Le #u Leu Leu Leu Leu  1               5  #                 10  #                 15Ala Leu Gly Pro Gly Val Gln Gly Cys Pro Se #r Gly Cys Gln Cys                 20  #                 25  #                 30Ser Gln Pro Gln Thr Val Phe Cys Thr Ala Ar #g Gln Gly Thr Thr                 35  #                 40  #                 45Val Pro Arg Asp Val Pro Pro Asp Thr Val Gl #y Leu Tyr Val Phe                 50  #                 55  #                 60Glu Asn Gly Ile Thr Met Leu Asp Ala Ser Se #r Phe Ala Gly Leu                 65  #                 70  #                 75Pro Gly Leu Gln Leu Leu Asp Leu Ser Gln As #n Gln Ile Ala Ser                 80  #                 85  #                 90Leu Arg Leu Pro Arg Leu Leu Leu Leu Asp Le #u Ser His Asn Ser                 95  #                100  #                105Leu Leu Ala Leu Glu Pro Gly Ile Leu Asp Th #r Ala Asn Val Glu                110   #               115   #               120Ala Leu Arg Leu Ala Gly Leu Gly Leu Gln Gl #n Leu Asp Glu Gly                125   #               130   #               135Leu Phe Ser Arg Leu Arg Asn Leu His Asp Le #u Asp Val Ser Asp                140   #               145   #               150Asn Gln Leu Glu Arg Val Pro Pro Val Ile Ar #g Gly Leu Arg Gly                155   #               160   #               165Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Ar #g Ile Ala Gln Leu                170   #               175   #               180Arg Pro Glu Asp Leu Ala Gly Leu Ala Ala Le #u Gln Glu Leu Asp                185   #               190   #               195Val Ser Asn Leu Ser Leu Gln Ala Leu Pro Gl #y Asp Leu Ser Gly                200   #               205   #               210Leu Phe Pro Arg Leu Arg Leu Leu Ala Ala Al #a Arg Asn Pro Phe                215   #               220   #               225Asn Cys Val Cys Pro Leu Ser Trp Phe Gly Pr #o Trp Val Arg Glu                230   #               235   #               240Ser His Val Thr Leu Ala Ser Pro Glu Glu Th #r Arg Cys His Phe                245   #               250   #               255Pro Pro Lys Asn Ala Gly Arg Leu Leu Leu Gl #u Leu Asp Tyr Ala                260   #               265   #               270Asp Phe Gly Cys Pro Ala Thr Thr Thr Thr Al #a Thr Val Pro Thr                275   #               280   #               285Thr Arg Pro Val Val Arg Glu Pro Thr Ala Le #u Ser Ser Ser Leu                290   #               295   #               300Ala Pro Thr Trp Leu Ser Pro Thr Ala Pro Al #a Thr Glu Ala Pro                305   #               310   #               315Ser Pro Pro Ser Thr Ala Pro Pro Thr Val Gl #y Pro Val Pro Gln                320   #               325   #               330Pro Gln Asp Cys Pro Pro Ser Thr Cys Leu As #n Gly Gly Thr Cys                335   #               340   #               345His Leu Gly Thr Arg His His Leu Ala Cys Le #u Cys Pro Glu Gly                350   #               355   #               360Phe Thr Gly Leu Tyr Cys Glu Ser Gln Met Gl #y Gln Gly Thr Arg                365   #               370   #               375Pro Ser Pro Thr Pro Val Thr Pro Arg Pro Pr #o Arg Ser Leu Thr                380   #               385   #               390Leu Gly Ile Glu Pro Val Ser Pro Thr Ser Le #u Arg Val Gly Leu                395   #               400   #               405Gln Arg Tyr Leu Gln Gly Ser Ser Val Gln Le #u Arg Ser Leu Arg                410   #               415   #               420Leu Thr Tyr Arg Asn Leu Ser Gly Pro Asp Ly #s Arg Leu Val Thr                425   #               430   #               435Leu Arg Leu Pro Ala Ser Leu Ala Glu Tyr Th #r Val Thr Gln Leu                440   #               445   #               450Arg Pro Asn Ala Thr Tyr Ser Val Cys Val Me #t Pro Leu Gly Pro                455   #               460   #               465Gly Arg Val Pro Glu Gly Glu Glu Ala Cys Gl #y Glu Ala His Thr                470   #               475   #               480Pro Pro Ala Val His Ser Asn His Ala Pro Va #l Thr Gln Ala Arg                485   #               490   #               495Glu Gly Asn Leu Pro Leu Leu Ile Ala Pro Al #a Leu Ala Ala Val                500   #               505   #               510Leu Leu Ala Ala Leu Ala Ala Val Gly Ala Al #a Tyr Cys Val Arg                515   #               520   #               525Arg Gly Arg Ala Met Ala Ala Ala Ala Gln As #p Lys Gly Gln Val                530   #               535   #               540Gly Pro Gly Ala Gly Pro Leu Glu Leu Glu Gl #y Val Lys Val Pro                545   #               550   #               555Leu Glu Pro Gly Pro Lys Ala Thr Glu Gly Gl #y Gly Glu Ala Leu                560   #               565   #               570Pro Ser Gly Ser Glu Cys Glu Val Pro Leu Me #t Gly Phe Pro Gly                575   #               580   #               585Pro Gly Leu Gln Ser Pro Leu His Ala Lys Pr #o Tyr Ile                590   #               595 <210> SEQ ID NO 70<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 70 ccctccactg ccccaccgac tg           #                   #                 22 <210> SEQ ID NO 71<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 71 cggttctggg gacgttaggg ctcg          #                   #                24 <210> SEQ ID NO 72<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 72 ctgcccaccg tccacctgcc tcaat          #                   #               25 <210> SEQ ID NO 73<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 73aggactgccc accgtccacc tgcctcaatg ggggcacatg ccacc    #                  #45 <210> SEQ ID NO 74 <211> LENGTH: 45 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide Pr #obe<400> SEQUENCE: 74 acgcaaagcc ctacatctaa gccagagaga gacagggcag ctggg   #                   #45 <210> SEQ ID NO 75 <211> LENGTH: 1077<212> TYPE: DNA <213> ORGANISM: Homo Sapien <400> SEQUENCE: 75ggcactagga caaccttctt cccttctgca ccactgcccg tacccttacc  #              50cgccccgcca cctccttgct accccactct tgaaaccaca gctgttggca  #             100gggtccccag ctcatgccag cctcatctcc tttcttgcta gcccccaaag  #             150ggcctccagg caacatgggg ggcccagtca gagagccggc actctcagtt  #             200gccctctggt tgagttgggg ggcagctctg ggggccgtgg cttgtgccat  #             250ggctctgctg acccaacaaa cagagctgca gagcctcagg agagaggtga  #             300gccggctgca ggggacagga ggcccctccc agaatgggga agggtatccc  #             350tggcagagtc tcccggagca gagttccgat gccctggaag cctgggagaa  #             400tggggagaga tcccggaaaa ggagagcagt gctcacccaa aaacagaaga  #             450agcagcactc tgtcctgcac ctggttccca ttaacgccac ctccaaggat  #             500gactccgatg tgacagaggt gatgtggcaa ccagctctta ggcgtgggag  #             550aggcctacag gcccaaggat atggtgtccg aatccaggat gctggagttt  #             600atctgctgta tagccaggtc ctgtttcaag acgtgacttt caccatgggt  #             650caggtggtgt ctcgagaagg ccaaggaagg caggagactc tattccgatg  #             700tataagaagt atgccctccc acccggaccg ggcctacaac agctgctata  #             750gcgcaggtgt cttccattta caccaagggg atattctgag tgtcataatt  #             800ccccgggcaa gggcgaaact taacctctct ccacatggaa ccttcctggg  #             850gtttgtgaaa ctgtgattgt gttataaaaa gtggctccca gcttggaaga  #             900ccagggtggg tacatactgg agacagccaa gagctgagta tataaaggag  #             950agggaatgtg caggaacaga ggcatcttcc tgggtttggc tccccgttcc  #            1000tcacttttcc cttttcattc ccacccccta gactttgatt ttacggatat  #            1050 cttgcttctg ttccccatgg agctccg          #                   #           1077 <210> SEQ ID NO 76<211> LENGTH: 250 <212> TYPE: PRT <213> ORGANISM: Homo Sapien<400> SEQUENCE: 76 Met Pro Ala Ser Ser Pro Phe Leu Leu Ala Pr#o Lys Gly Pro Pro   1               5  #                 10 #                 15 Gly Asn Met Gly Gly Pro Val Arg Glu Pro Al#a Leu Ser Val Ala                  20  #                 25 #                 30 Leu Trp Leu Ser Trp Gly Ala Ala Leu Gly Al#a Val Ala Cys Ala                  35  #                 40 #                 45 Met Ala Leu Leu Thr Gln Gln Thr Glu Leu Gl#n Ser Leu Arg Arg                  50  #                 55 #                 60 Glu Val Ser Arg Leu Gln Gly Thr Gly Gly Pr#o Ser Gln Asn Gly                  65  #                 70 #                 75 Glu Gly Tyr Pro Trp Gln Ser Leu Pro Glu Gl#n Ser Ser Asp Ala                  80  #                 85 #                 90 Leu Glu Ala Trp Glu Asn Gly Glu Arg Ser Ar#g Lys Arg Arg Ala                  95  #                100 #                105 Val Leu Thr Gln Lys Gln Lys Lys Gln His Se#r Val Leu His Leu                 110   #               115  #               120 Val Pro Ile Asn Ala Thr Ser Lys Asp Asp Se#r Asp Val Thr Glu                 125   #               130  #               135 Val Met Trp Gln Pro Ala Leu Arg Arg Gly Ar#g Gly Leu Gln Ala                 140   #               145  #               150 Gln Gly Tyr Gly Val Arg Ile Gln Asp Ala Gl#y Val Tyr Leu Leu                 155   #               160  #               165 Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Ph#e Thr Met Gly Gln                 170   #               175  #               180 Val Val Ser Arg Glu Gly Gln Gly Arg Gln Gl#u Thr Leu Phe Arg                 185   #               190  #               195 Cys Ile Arg Ser Met Pro Ser His Pro Asp Ar#g Ala Tyr Asn Ser                 200   #               205  #               210 Cys Tyr Ser Ala Gly Val Phe His Leu His Gl#n Gly Asp Ile Leu                 215   #               220  #               225 Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Le#u Asn Leu Ser Pro                 230   #               235  #               240 His Gly Thr Phe Leu Gly Phe Val Lys Leu                245   #               250 <210> SEQ ID NO 77<211> LENGTH: 2849 <212> TYPE: DNA <213> ORGANISM: Homo Sapien<400> SEQUENCE: 77cactttctcc ctctcttcct ttactttcga gaaaccgcgc ttccgcttct  #              50ggtcgcagag acctcggaga ccgcgccggg gagacggagg tgctgtgggt  #             100gggggggacc tgtggctgct cgtaccgccc cccaccctcc tcttctgcac  #             150tgccgtcctc cggaagacct tttcccctgc tctgtttcct tcaccgagtc  #             200tgtgcatcgc cccggacctg gccgggagga ggcttggccg gcgggagatg  #             250ctctaggggc ggcgcgggag gagcggccgg cgggacggag ggcccggcag  #             300gaagatgggc tcccgtggac agggactctt gctggcgtac tgcctgctcc  #             350ttgcctttgc ctctggcctg gtcctgagtc gtgtgcccca tgtccagggg  #             400gaacagcagg agtgggaggg gactgaggag ctgccgtcgc ctccggacca  #             450tgccgagagg gctgaagaac aacatgaaaa atacaggccc agtcaggacc  #             500aggggctccc tgcttcccgg tgcttgcgct gctgtgaccc cggtacctcc  #             550atgtacccgg cgaccgccgt gccccagatc aacatcacta tcttgaaagg  #             600ggagaagggt gaccgcggag atcgaggcct ccaagggaaa tatggcaaaa  #             650caggctcagc aggggccagg ggccacactg gacccaaagg gcagaagggc  #             700tccatggggg cccctgggga gcggtgcaag agccactacg ccgccttttc  #             750ggtgggccgg aagaagccca tgcacagcaa ccactactac cagacggtga  #             800tcttcgacac ggagttcgtg aacctctacg accacttcaa catgttcacc  #             850ggcaagttct actgctacgt gcccggcctc tacttcttca gcctcaacgt  #             900gcacacctgg aaccagaagg agacctacct gcacatcatg aagaacgagg  #             950aggaggtggt gatcttgttc gcgcaggtgg gcgaccgcag catcatgcaa  #            1000agccagagcc tgatgctgga gctgcgagag caggaccagg tgtgggtacg  #            1050cctctacaag ggcgaacgtg agaacgccat cttcagcgag gagctggaca  #            1100cctacatcac cttcagtggc tacctggtca agcacgccac cgagccctag  #            1150ctggccggcc acctcctttc ctctcgccac cttccacccc tgcgctgtgc  #            1200tgaccccacc gcctcttccc cgatccctgg actccgactc cctggctttg  #            1250gcattcagtg agacgccctg cacacacaga aagccaaagc gatcggtgct  #            1300cccagatccc gcagcctctg gagagagctg acggcagatg aaatcaccag  #            1350ggcggggcac ccgcgagaac cctctgggac cttccgcggc cctctctgca  #            1400cacatcctca agtgaccccg cacggcgaga cgcgggtggc ggcagggcgt  #            1450cccagggtgc ggcaccgcgg ctccagtcct tggaaataat taggcaaatt  #            1500ctaaaggtct caaaaggagc aaagtaaacc gtggaggaca aagaaaaggg  #            1550ttgttatttt tgtctttcca gccagcctgc tggctcccaa gagagaggcc  #            1600ttttcagttg agactctgct taagagaaga tccaaagtta aagctctggg  #            1650gtcaggggag gggccggggg caggaaacta cctctggctt aattctttta  #            1700agccacgtag gaactttctt gagggatagg tggaccctga catccctgtg  #            1750gccttgccca agggctctgc tggtctttct gagtcacagc tgcgaggtga  #            1800tgggggctgg ggccccaggc gtcagcctcc cagagggaca gctgagcccc  #            1850ctgccttggc tccaggttgg tagaagcagc cgaagggctc ctgacagtgg  #            1900ccagggaccc ctgggtcccc caggcctgca gatgtttcta tgaggggcag  #            1950agctccttgg tacatccatg tgtggctctg ctccacccct gtgccacccc  #            2000agagccctgg ggggtggtct ccatgcctgc caccctggca tcggctttct  #            2050gtgccgcctc ccacacaaat cagccccaga aggccccggg gccttggctt  #            2100ctgtttttta taaaacacct caagcagcac tgcagtctcc catctcctcg  #            2150tgggctaagc atcaccgctt ccacgtgtgt tgtgttggtt ggcagcaagg  #            2200ctgatccaga ccccttctgc ccccactgcc ctcatccagg cctctgacca  #            2250gtagcctgag aggggctttt tctaggcttc agagcagggg agagctggaa  #            2300ggggctagaa agctcccgct tgtctgtttc tcaggctcct gtgagcctca  #            2350gtcctgagac cagagtcaag aggaagtaca cgtcccaatc acccgtgtca  #            2400ggattcactc tcaggagctg ggtggcagga gaggcaatag cccctgtggc  #            2450aattgcagga ccagctggag cagggttgcg gtgtctccac ggtgctctcg  #            2500ccctgcccat ggccacccca gactctgatc tccaggaacc ccatagcccc  #            2550tctccacctc accccatgtt gatgcccagg gtcactcttg ctacccgctg  #            2600ggcccccaaa cccccgctgc ctctcttcct tccccccatc ccccacctgg  #            2650ttttgactaa tcctgcttcc ctctctgggc ctggctgccg ggatctgggg  #            2700tccctaagtc cctctcttta aagaacttct gcgggtcaga ctctgaagcc  #            2750gagttgctgt gggcgtgccc ggaagcagag cgccacactc gctgcttaag  #            2800ctcccccagc tctttccaga aaacattaaa ctcagaattg tgttttcaa  #            2849 <210> SEQ ID NO 78 <211> LENGTH: 281 <212> TYPE: PRT<213> ORGANISM: Homo Sapien <400> SEQUENCE: 78Met Gly Ser Arg Gly Gln Gly Leu Leu Leu Al #a Tyr Cys Leu Leu  1               5  #                 10  #                 15Leu Ala Phe Ala Ser Gly Leu Val Leu Ser Ar #g Val Pro His Val                 20  #                 25  #                 30Gln Gly Glu Gln Gln Glu Trp Glu Gly Thr Gl #u Glu Leu Pro Ser                 35  #                 40  #                 45Pro Pro Asp His Ala Glu Arg Ala Glu Glu Gl #n His Glu Lys Tyr                 50  #                 55  #                 60Arg Pro Ser Gln Asp Gln Gly Leu Pro Ala Se #r Arg Cys Leu Arg                 65  #                 70  #                 75Cys Cys Asp Pro Gly Thr Ser Met Tyr Pro Al #a Thr Ala Val Pro                 80  #                 85  #                 90Gln Ile Asn Ile Thr Ile Leu Lys Gly Glu Ly #s Gly Asp Arg Gly                 95  #                100  #                105Asp Arg Gly Leu Gln Gly Lys Tyr Gly Lys Th #r Gly Ser Ala Gly                110   #               115   #               120Ala Arg Gly His Thr Gly Pro Lys Gly Gln Ly #s Gly Ser Met Gly                125   #               130   #               135Ala Pro Gly Glu Arg Cys Lys Ser His Tyr Al #a Ala Phe Ser Val                140   #               145   #               150Gly Arg Lys Lys Pro Met His Ser Asn His Ty #r Tyr Gln Thr Val                155   #               160   #               165Ile Phe Asp Thr Glu Phe Val Asn Leu Tyr As #p His Phe Asn Met                170   #               175   #               180Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gl #y Leu Tyr Phe Phe                185   #               190   #               195Ser Leu Asn Val His Thr Trp Asn Gln Lys Gl #u Thr Tyr Leu His                200   #               205   #               210Ile Met Lys Asn Glu Glu Glu Val Val Ile Le #u Phe Ala Gln Val                215   #               220   #               225Gly Asp Arg Ser Ile Met Gln Ser Gln Ser Le #u Met Leu Glu Leu                230   #               235   #               240Arg Glu Gln Asp Gln Val Trp Val Arg Leu Ty #r Lys Gly Glu Arg                245   #               250   #               255Glu Asn Ala Ile Phe Ser Glu Glu Leu Asp Th #r Tyr Ile Thr Phe                260   #               265   #               270Ser Gly Tyr Leu Val Lys His Ala Thr Glu Pr #o                 275  #               280 <210> SEQ ID NO 79 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 79 tacaggccca gtcaggacca gggg          #                   #                24 <210> SEQ ID NO 80<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 80 ctgaagaagt agaggccggg cacg          #                   #                24 <210> SEQ ID NO 81<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 81cccggtgctt gcgctgctgt gaccccggta cctccatgta cccgg    #                  #45 <210> SEQ ID NO 82 <211> LENGTH: 2284 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 82gcggagcatc cgctgcggtc ctcgccgaga cccccgcgcg gattcgccgg  #              50tccttcccgc gggcgcgaca gagctgtcct cgcacctgga tggcagcagg  #             100ggcgccgggg tcctctcgac gccagagaga aatctcatca tctgtgcagc  #             150cttcttaaag caaactaaga ccagagggag gattatcctt gacctttgaa  #             200gaccaaaact aaactgaaat ttaaaatgtt cttcggggga gaagggagct  #             250tgacttacac tttggtaata atttgcttcc tgacactaag gctgtctgct  #             300agtcagaatt gcctcaaaaa gagtctagaa gatgttgtca ttgacatcca  #             350gtcatctctt tctaagggaa tcagaggcaa tgagcccgta tatacttcaa  #             400ctcaagaaga ctgcattaat tcttgctgtt caacaaaaaa catatcaggg  #             450gacaaagcat gtaacttgat gatcttcgac actcgaaaaa cagctagaca  #             500acccaactgc tacctatttt tctgtcccaa cgaggaagcc tgtccattga  #             550aaccagcaaa aggacttatg agttacagga taattacaga ttttccatct  #             600ttgaccagaa atttgccaag ccaagagtta ccccaggaag attctctctt  #             650acatggccaa ttttcacaag cagtcactcc cctagcccat catcacacag  #             700attattcaaa gcccaccgat atctcatgga gagacacact ttctcagaag  #             750tttggatcct cagatcacct ggagaaacta tttaagatgg atgaagcaag  #             800tgcccagctc cttgcttata aggaaaaagg ccattctcag agttcacaat  #             850tttcctctga tcaagaaata gctcatctgc tgcctgaaaa tgtgagtgcg  #             900ctcccagcta cggtggcagt tgcttctcca cataccacct cggctactcc  #             950aaagcccgcc acccttctac ccaccaatgc ttcagtgaca ccttctggga  #            1000cttcccagcc acagctggcc accacagctc cacctgtaac cactgtcact  #            1050tctcagcctc ccacgaccct catttctaca gtttttacac gggctgcggc  #            1100tacactccaa gcaatggcta caacagcagt tctgactacc acctttcagg  #            1150cacctacgga ctcgaaaggc agcttagaaa ccataccgtt tacagaaatc  #            1200tccaacttaa ctttgaacac agggaatgtg tataacccta ctgcactttc  #            1250tatgtcaaat gtggagtctt ccactatgaa taaaactgct tcctgggaag  #            1300gtagggaggc cagtccaggc agttcctccc agggcagtgt tccagaaaat  #            1350cagtacggcc ttccatttga aaaatggctt cttatcgggt ccctgctctt  #            1400tggtgtcctg ttcctggtga taggcctcgt cctcctgggt agaatccttt  #            1450cggaatcact ccgcaggaaa cgttactcaa gactggatta tttgatcaat  #            1500gggatctatg tggacatcta aggatggaac tcggtgtctc ttaattcatt  #            1550tagtaaccag aagcccaaat gcaatgagtt tctgctgact tgctagtctt  #            1600agcaggaggt tgtattttga agacaggaaa atgccccctt ctgctttcct  #            1650tttttttttt ggagacagag tcttgctctg ttgcccaggc tggagtgcag  #            1700tagcacgatc tcggctctca ccgcaacctc cgtctcctgg gttcaagcga  #            1750ttctcctgcc tcagcctcct aagtatctgg gattacaggc atgtgccacc  #            1800acacctgggt gatttttgta tttttagtag agacggggtt tcaccatgtt  #            1850ggtcaggctg gtctcaaact cctgacctag tgatccaccc tcctcggcct  #            1900cccaaagtgc tgggattaca ggcatgagcc accacagctg gcccccttct  #            1950gttttatgtt tggtttttga gaaggaatga agtgggaacc aaattaggta  #            2000attttgggta atctgtctct aaaatattag ctaaaaacaa agctctatgt  #            2050aaagtaataa agtataattg ccatataaat ttcaaaattc aactggcttt  #            2100tatgcaaaga aacaggttag gacatctagg ttccaattca ttcacattct  #            2150tggttccaga taaaatcaac tgtttatatc aatttctaat ggatttgctt  #            2200ttctttttat atggattcct ttaaaactta ttccagatgt agttccttcc  #            2250 aattaaatat ttgaataaat cttttgttac tcaa       #                   #      2284 <210> SEQ ID NO 83 <211> LENGTH: 431<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 83Met Phe Phe Gly Gly Glu Gly Ser Leu Thr Ty #r Thr Leu Val Ile  1               5  #                 10  #                 15Ile Cys Phe Leu Thr Leu Arg Leu Ser Ala Se #r Gln Asn Cys Leu                 20  #                 25  #                 30Lys Lys Ser Leu Glu Asp Val Val Ile Asp Il #e Gln Ser Ser Leu                 35  #                 40  #                 45Ser Lys Gly Ile Arg Gly Asn Glu Pro Val Ty #r Thr Ser Thr Gln                 50  #                 55  #                 60Glu Asp Cys Ile Asn Ser Cys Cys Ser Thr Ly #s Asn Ile Ser Gly                 65  #                 70  #                 75Asp Lys Ala Cys Asn Leu Met Ile Phe Asp Th #r Arg Lys Thr Ala                 80  #                 85  #                 90Arg Gln Pro Asn Cys Tyr Leu Phe Phe Cys Pr #o Asn Glu Glu Ala                 95  #                100  #                105Cys Pro Leu Lys Pro Ala Lys Gly Leu Met Se #r Tyr Arg Ile Ile                110   #               115   #               120Thr Asp Phe Pro Ser Leu Thr Arg Asn Leu Pr #o Ser Gln Glu Leu                125   #               130   #               135Pro Gln Glu Asp Ser Leu Leu His Gly Gln Ph #e Ser Gln Ala Val                140   #               145   #               150Thr Pro Leu Ala His His His Thr Asp Tyr Se #r Lys Pro Thr Asp                155   #               160   #               165Ile Ser Trp Arg Asp Thr Leu Ser Gln Lys Ph #e Gly Ser Ser Asp                170   #               175   #               180His Leu Glu Lys Leu Phe Lys Met Asp Glu Al #a Ser Ala Gln Leu                185   #               190   #               195Leu Ala Tyr Lys Glu Lys Gly His Ser Gln Se #r Ser Gln Phe Ser                200   #               205   #               210Ser Asp Gln Glu Ile Ala His Leu Leu Pro Gl #u Asn Val Ser Ala                215   #               220   #               225Leu Pro Ala Thr Val Ala Val Ala Ser Pro Hi #s Thr Thr Ser Ala                230   #               235   #               240Thr Pro Lys Pro Ala Thr Leu Leu Pro Thr As #n Ala Ser Val Thr                245   #               250   #               255Pro Ser Gly Thr Ser Gln Pro Gln Leu Ala Th #r Thr Ala Pro Pro                260   #               265   #               270Val Thr Thr Val Thr Ser Gln Pro Pro Thr Th #r Leu Ile Ser Thr                275   #               280   #               285Val Phe Thr Arg Ala Ala Ala Thr Leu Gln Al #a Met Ala Thr Thr                290   #               295   #               300Ala Val Leu Thr Thr Thr Phe Gln Ala Pro Th #r Asp Ser Lys Gly                305   #               310   #               315Ser Leu Glu Thr Ile Pro Phe Thr Glu Ile Se #r Asn Leu Thr Leu                320   #               325   #               330Asn Thr Gly Asn Val Tyr Asn Pro Thr Ala Le #u Ser Met Ser Asn                335   #               340   #               345Val Glu Ser Ser Thr Met Asn Lys Thr Ala Se #r Trp Glu Gly Arg                350   #               355   #               360Glu Ala Ser Pro Gly Ser Ser Ser Gln Gly Se #r Val Pro Glu Asn                365   #               370   #               375Gln Tyr Gly Leu Pro Phe Glu Lys Trp Leu Le #u Ile Gly Ser Leu                380   #               385   #               390Leu Phe Gly Val Leu Phe Leu Val Ile Gly Le #u Val Leu Leu Gly                395   #               400   #               405Arg Ile Leu Ser Glu Ser Leu Arg Arg Lys Ar #g Tyr Ser Arg Leu                410   #               415   #               420Asp Tyr Leu Ile Asn Gly Ile Tyr Val Asp Il #e                 425  #               430 <210> SEQ ID NO 84 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 84 agggaggatt atccttgacc tttgaagacc         #                   #           30 <210> SEQ ID NO 85 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 85 gaagcaagtg cccagctc              #                  #                   #  18 <210> SEQ ID NO 86 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 86 cgggtccctg ctctttgg              #                  #                   #  18 <210> SEQ ID NO 87 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 87 caccgtagct gggagcgcac tcac          #                   #                24 <210> SEQ ID NO 88<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 88 agtgtaagtc aagctccc             #                   #                   #  18 <210> SEQ ID NO 89<211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 89gcttcctgac actaaggctg tctgctagtc agaattgcct caaaaagag  #               49 <210> SEQ ID NO 90 <211> LENGTH: 957 <212> TYPE: DNA<213> ORGANISM: Homo Sapien <400> SEQUENCE: 90cctggaagat gcgcccattg gctggtggcc tgctcaaggt ggtgttcgtg  #              50gtcttcgcct ccttgtgtgc ctggtattcg gggtacctgc tcgcagagct  #             100cattccagat gcacccctgt ccagtgctgc ctatagcatc cgcagcatcg  #             150gggagaggcc tgtcctcaaa gctccagtcc ccaaaaggca aaaatgtgac  #             200cactggactc cctgcccatc tgacacctat gcctacaggt tactcagcgg  #             250aggtggcaga agcaagtacg ccaaaatctg ctttgaggat aacctactta  #             300tgggagaaca gctgggaaat gttgccagag gaataaacat tgccattgtc  #             350aactatgtaa ctgggaatgt gacagcaaca cgatgttttg atatgtatga  #             400aggcgataac tctggaccga tgacaaagtt tattcagagt gctgctccaa  #             450aatccctgct cttcatggtg acctatgacg acggaagcac aagactgaat  #             500aacgatgcca agaatgccat agaagcactt ggaagtaaag aaatcaggaa  #             550catgaaattc aggtctagct gggtatttat tgcagcaaaa ggcttggaac  #             600tcccttccga aattcagaga gaaaagatca accactctga tgctaagaac  #             650aacagatatt ctggctggcc tgcagagatc cagatagaag gctgcatacc  #             700caaagaacga agctgacact gcagggtcct gagtaaatgt gttctgtata  #             750aacaaatgca gctggaatcg ctcaagaatc ttatttttct aaatccaaca  #             800gcccatattt gatgagtatt ttgggtttgt tgtaaaccaa tgaacatttg  #             850ctagttgtat caaatcttgg tacgcagtat ttttatacca gtattttatg  #             900tagtgaagat gtcaattagc aggaaactaa aatgaatgga aattcttaaa  #             950 aaaaaaa                  #                  #                   #         957 <210> SEQ ID NO 91 <211> LENGTH: 235<212> TYPE: PRT <213> ORGANISM: Homo Sapien <400> SEQUENCE: 91Met Arg Pro Leu Ala Gly Gly Leu Leu Lys Va #l Val Phe Val Val  1               5  #                 10  #                 15Phe Ala Ser Leu Cys Ala Trp Tyr Ser Gly Ty #r Leu Leu Ala Glu                 20  #                 25  #                 30Leu Ile Pro Asp Ala Pro Leu Ser Ser Ala Al #a Tyr Ser Ile Arg                 35  #                 40  #                 45Ser Ile Gly Glu Arg Pro Val Leu Lys Ala Pr #o Val Pro Lys Arg                 50  #                 55  #                 60Gln Lys Cys Asp His Trp Thr Pro Cys Pro Se #r Asp Thr Tyr Ala                 65  #                 70  #                 75Tyr Arg Leu Leu Ser Gly Gly Gly Arg Ser Ly #s Tyr Ala Lys Ile                 80  #                 85  #                 90Cys Phe Glu Asp Asn Leu Leu Met Gly Glu Gl #n Leu Gly Asn Val                 95  #                100  #                105Ala Arg Gly Ile Asn Ile Ala Ile Val Asn Ty #r Val Thr Gly Asn                110   #               115   #               120Val Thr Ala Thr Arg Cys Phe Asp Met Tyr Gl #u Gly Asp Asn Ser                125   #               130   #               135Gly Pro Met Thr Lys Phe Ile Gln Ser Ala Al #a Pro Lys Ser Leu                140   #               145   #               150Leu Phe Met Val Thr Tyr Asp Asp Gly Ser Th #r Arg Leu Asn Asn                155   #               160   #               165Asp Ala Lys Asn Ala Ile Glu Ala Leu Gly Se #r Lys Glu Ile Arg                170   #               175   #               180Asn Met Lys Phe Arg Ser Ser Trp Val Phe Il #e Ala Ala Lys Gly                185   #               190   #               195Leu Glu Leu Pro Ser Glu Ile Gln Arg Glu Ly #s Ile Asn His Ser                200   #               205   #               210Asp Ala Lys Asn Asn Arg Tyr Ser Gly Trp Pr #o Ala Glu Ile Gln                215   #               220   #               225Ile Glu Gly Cys Ile Pro Lys Glu Arg Ser                 230  #               235 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 92 aatgtgacca ctggactccc             #                  #                   # 20 <210> SEQ ID NO 93 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 93 aggcttggaa ctcccttc              #                  #                   #  18 <210> SEQ ID NO 94 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 94 aagattcttg agcgattcca gctg          #                   #                24 <210> SEQ ID NO 95<211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 95aatccctgct cttcatggtg acctatgacg acggaagcac aagactg   #                47 <210> SEQ ID NO 96 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 96 ctcaagaagc acgcgtactg c           #                   #                   #21 <210> SEQ ID NO 97<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 97 ccaacctcag cttccgcctc tacga          #                   #               25 <210> SEQ ID NO 98<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 98 catccaggct cgccactg             #                   #                   #  18 <210> SEQ ID NO 99<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 99 tggcaaggaa tgggaacagt            #                   #                   # 20 <210> SEQ ID NO 100<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 100 atgctgccag acctgatcgc agaca          #                   #               25 <210> SEQ ID NO 101<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 101 gggcagaaat ccagccact             #                   #                   # 19 <210> SEQ ID NO 102<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 102 cccttcgcct gcttttga             #                   #                   #  18 <210> SEQ ID NO 103<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 103 gccatctaat tgaagcccat cttccca          #                   #             27 <210> SEQ ID NO 104<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 104 ctggcggtgt cctctcctt             #                   #                   # 19 <210> SEQ ID NO 105<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 105 cctcggtctc ctcatctgtg a           #                   #                   #21 <210> SEQ ID NO 106<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 106 tggcccagct gacgagccct            #                   #                   # 20 <210> SEQ ID NO 107<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 107 ctcataggca ctcggttctg g           #                   #                   #21 <210> SEQ ID NO 108<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 108 tggctcccag cttggaaga             #                   #                   # 19 <210> SEQ ID NO 109<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 109 cagctcttgg ctgtctccag tatgtaccca         #                   #           30 <210> SEQ ID NO 110 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 110 gatgcctctg ttcctgcaca t           #                   #                   #21 <210> SEQ ID NO 111<211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide pr#obe <400> SEQUENCE: 111ggattctaat acgactcact atagggctgc ccgcaacccc ttcaactg  #                48 <210> SEQ ID NO 112 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 112ctatgaaatt aaccctcact aaagggaccg cagctgggtg accgtgta  #                48 <210> SEQ ID NO 113 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 113 ggattctaat acgactcact atagggccgc cccgccacct cct    #                   # 43 <210> SEQ ID NO 114 <211> LENGTH: 48<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 114ctatgaaatt aaccctcact aaagggactc gagacaccac ctgaccca  #                48 <210> SEQ ID NO 115 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 115ggattctaat acgactcact atagggccca aggaaggcag gagactct  #                48 <210> SEQ ID NO 116 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic Oligonucleotide pr #obe<400> SEQUENCE: 116ctatgaaatt aaccctcact aaagggacta gggggtggga atgaaaag  #                48 <210> SEQ ID NO 117 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 117ggattctaat acgactcact atagggcccc cctgagctct cccgtgta  #                48 <210> SEQ ID NO 118 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 118ctatgaaatt aaccctcact aaagggaagg ctcgccactg gtcgtaga  #                48 <210> SEQ ID NO 119 <211> LENGTH: 48 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 119ggattctaat acgactcact atagggcaag gagccgggac ccaggaga  #                48 <210> SEQ ID NO 120 <211> LENGTH: 47 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Synthetic oligonucleotide pr #obe<400> SEQUENCE: 120ctatgaaatt aaccctcact aaagggaggg ggcccttggt gctgagt   #                47

What is claimed is:
 1. An antibody that binds to the polypeptide shownin FIG, 2 (SEQ ID NO:2).
 2. The antibody of claim 1, which is amonoclonal antibody.
 3. The antibody of claim 1, which is a humanizedantibody.
 4. An antigen binding fragment of the antibody of claim
 1. 5.The antibody of claim 1 which is labeled.